General Introduction to In Situ Hybridization

General Introduction toIn Situ Hybridization

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1In situ hybridization techniques allowspecific nucleic acid sequences to bedetected in morphologically preservedchromosomes, cells or tissue sections. Incombination with immunocytochemistry,in situ hybridization can relate microscopictopological information to gene activity atthe DN A, mRN A, and protein level.

The technique was originally developed byPardue and Gall (1969) and (independ-ently) by John et al. (1969). At this timeradioisotopes were the only labels availablefor nucleic acids, and autoradiography wasthe only means of detecting hybridizedsequences. Furthermore, as molecular clon-ing was not possible in those days, in situhybridization was restricted to those sequences that could be purified and isolatedby conventional biochemical methods (e.g.,mouse satellite DN A, viral DN A, ribosomalRN As).

Molecular cloning of nucleic acids andimproved radiolabeling techniques havechanged this picture dramatically. Forexample, DN A sequences a few hundredbase pairs long can be detected in meta-phase chromosomes by autoradiography(H arper et al., 1981; Jhanwag et al., 1984;Rabin et al., 1984; Schroeder et al., 1984).Also radioactive in situ techniques can detectlow copy number mRN A molecules inindividual cells (H arper et al., 1986). Someyears ago, chemically synthesized, radio-actively labeled oligonucleotides began to beused, especially for in situ mRN A detection(Coghlan et al., 1985).

In spite of the high sensitivity and wideapplicability of in situ hybridization tech-niques, their use has been limited to researchlaboratories. This is probably due to theproblems associated with radioactiveprobes, such as the safety measures required,limited shelf life, and extensive time requiredfor autoradiography. In addition, the scatterinherent in radioactive decay limits thespatial resolution of the technique.

H owever, preparing nucleic acid probeswith a stable nonradioactive label removesthe major obstacles which hinder the generalapplication of in situ hybridization. Further-more, it opens new opportunities for com-bining different labels in one experiment.The many sensitive antibody detectionsystems available for such probes furtherenhances the flexibility of this method. Inthis manual, therefore, we describe nonradio-active alternatives for in situ hybridization.

General Introduction to In SituHybridization

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General Introduction to In Situ H ybridization

1A few years ago, the chemical synthesis of oligonucleotides containing functionalgroups (e.g., primary aliphatic amines orsulfhydryl groups) was described. Thesecan react with haptens, fluorochromes orenzymes to produce a stable probe whichcan be used for in situ hybridization exper-iments (Agrawal et al., 1986; Chollet andKawashima, 1985; H aralambidis et al.,1987; Jablonski et al., 1986). Modifiedoligonucleotides can also be obtained withthe DIG/Genius™ system (Mühlegger etal., 1990). Such oligonucleotide probes willundoubtedly be widely used as automatedoligonucleotide synthesis makes themavailable to researchers not familiar withDN A recombinant technology.

This manual concentrates on two labelingsystems:! Indirect methods using digoxigenin

(detected by specific antibodies) andbiotin (detected by streptavidin)

! Direct methods using fluorescein orother fluorochromes directly coupled tothe nucleotide

The ordering information in Chapter 7 listsall the kits and single reagents BoehringerMannheim offers for nonradioactivelabeling and detection.

Direct and indirect methodsThere are two types of nonradioactive hybridization methods: direct and indirect.In the direct method, the detectable mole-cule (reporter) is bound directly to thenucleic acid probe so that probe-targethybrids can be visualized under a micro-scope immediately after the hybridizationreaction. For such methods it is essential thatthe probe-reporter bond survives the ratherharsh hybridization and washing conditions.Perhaps more important, however, is, thatthe reporter molecule does not interferewith the hybridization reaction. Theterminal fluorochrome labeling procedure ofRN A probes developed by Bauman et al.(1980, 1984), and the direct enzyme labelingprocedure of nucleic acids described byRenz and Kurz (1984) meet these criteria.Boehringer Mannheim has introducedseveral fluorochrome-labeled nucleotidesthat can be used for labeling and directdetection of DN A or RN A probes.

If antibodies against the reporter moleculesare available, direct methods may also beconverted to indirect immunochemicalamplification methods (Bauman et al.,1981; Lansdorp et al., 1984; Pinkel et al.,1986).

Indirect procedures require the probe tocontain a reporter molecule, introducedchemically or enzymatically, that can bedetected by affinity cytochemistry. Again,the presence of the label should notinterfere with the hybridization reaction orthe stability of the resulting hybrid. Thereporter molecule should, however, beaccessible to antibodies. A number of suchhapten modifications has been described(Langer et al., 1981; Leary et al., 1983;Landegent et al., 1984; Tchen et al., 1984;H opman et al., 1986; H opman et al., 1987;Shroyer and N akane, 1983; Van Prooijen etal., 1982; Viscidi et al., 1986; Rudkin andStollar, 1977; Raap et al., 1989). O ne of themost popular is the biotin-streptavidinsystem. Boehringer Mannheim offers analternative, the Digoxigenin (DIG/Genius™)System which is described in detail later inthis chapter.

Guidelines forIn Situ Hybridization

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Guidelines for In SituHybridization

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An in situ hybridization protocol followsthis general outline:! Preparation of slides and fixation of

material ! Pretreatments of material on slides, e.g.,

permeabilization of cells and tissues! Denaturation of in situ target DN A (not

necessary for mRN A target)! Preparation of probe! In situ hybridization! Posthybridization washes! Immunocytochemistry! Microscopy

All these steps are discussed below indetail. The information provided will make it easier to decide whether a certain stepshould or should not be included in a givenin situ hybridization protocol.

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2Pretreatments of material on the slideEndogenous enzyme inactivationWhen an enzyme is used as the label, theendogenous enzyme activity may have to beinactivated. For peroxidase, this is done bytreating the sections with 1% H 2O 2 inmethanol for 30 min. For alkaline phos-phatase, levamisole may be added to thesubstrate solution, but this may be unnec-essary since residual alkaline phosphataseactivity is usually lost during hybridization.

RNase treatmentRN ase treatment serves to remove endo-genous RN A and may improve the signal-to-noise ratio in DN A-DN A hybridizations.This treatment can also be used as a control inhybridizations with (m)RN A as target. It isdone by incubating the preparations inDN ase-free RN ase (100 µg/ml) in 2x SSC at37°C for 60 min. (SSC = 150 mM N aCl,15 mM sodium citrate, pH 7.4).

HCl treatmentIn several protocols a 20–30 min treatmentwith 200 mM H Cl is included. The preciseaction of the acid is not known, butextraction of proteins and partial hydrolysisof the target sequences may contribute to animprovement in the signal-to-noise ratio.

Detergent treatmentPreparations may be pretreated with Triton®

X-100, sodium dodecyl sulfate, or otherdetergents if lipid membrane componentshave not been extracted by other proceduressuch as fixation, dehydration, embedding,and endogenous enzyme inactivationprocedures.

Slide preparationFor chromosome spreads, alcohol/ether(1:1) cleaned slides are sufficient. H owever,since tissue sections may be lost during theprocedure, use either polylysine orglutaraldehyde-activated gelatin chromealuminum slides for these sections.

FixationTo preserve morphology, the biologicalmaterial must be fixed. From a chemicalpoint of view, there is little limitation in thetype of fixation used because one of thefollowing will be true:! The functional groups involved in base

pairing are protected in the double helixstructure of duplex DN A.

! RN A is fairly unreactive to crosslinkingagents.

! The reaction is reversible (e.g., withformaldehyde).

For metaphase chromosome spreads, meth-anol/acetic acid fixation is usually sufficient.For paraffin-embedded tissue sections, useformalin fixation. Cryostat sections fixed for30 min with 4% formaldehyde or withBouin’s fixative have been used successfully,as well as paraformaldehyde vapor fixation.Tissues can also be freeze-dried.

It should be noted that the DN A and RN Atarget sequences are surrounded byproteins and that extensive crosslinking ofthese proteins masks the target nucleicacid. Therefore, permeabilization proce-dures are often required.

Unfortunately, a fixation protocol whichcan be used for all substrates has not yetbeen described. The fixation and pretreat-ment protocols must be optimized fordifferent applications.

Details of the Technique

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Denaturation of probe and targetFor in situ hybridization to chromosomalDN A, the DN A target must be denatured.In general such treatments may lead to lossof morphology, so in practice, a compro-mise must be found between hybridizationsignal and morphology. Alkaline denatura-tions have traditionally been used. H eatdenaturations have also become popular,because of their experimental simplicity andgreater effectiveness. Variations in time andtemperature should be evaluated to find thebest conditions for denaturation.

For heat denaturation, the probe and targetchromosomal DN A may be denaturedsimultaneously. To accomplish this, put theprobe on the slide and cover with acoverslip. Bring the slide to 80°C for 2 minin an oven for the denaturation and thencool to 37°C. For tissue sections, ifnecessary, extend the time of denaturationat 80°C to 10 min.

For competition in situ hybridization,where the labeled probe is allowed toreanneal with unlabeled competitor DN A,denature the chromosomal preparationand probe separately.

HybridizationSee Chapter 3 for the composition of thehybridization solution, and the factorsaffecting hybridization kinetics and hybridstability.

Protease treatmentProtease treatment serves to increase targetaccessibility by digesting the protein thatsurrounds the target nucleic acid. To digestthe sample, incubate the preparations withup to 500 µg/ml Proteinase K (the optimalamount must be determined) in 20 mMTris-H Cl, 2 mM CaCl2, pH 7.4, for 7.5–30min at 37°C. For example, with chromo-somes or isolated preparations of nuclei, a reasonable starting point for proteasedigestion is up to 1 µg/ml Proteinase K for 7.5 min. For formalin-fixed material,5–15 µg/ml for 15–30 min usually givesgood results.

The use of pepsin has been shown to giveexcellent results for formalin-fixed, paraffin-embedded tissue sections. Routine pepsindigestion involves incubating the prepara-tions for 30 min at 37°C in 200 mM H Clcontaining 500 µg/ml pepsin. For thepretreatment of chromosome spreads withpepsin see page 70.

O ther hydrolases, such as Collagenase anddiastase, may be tried if preparations ofconnective tissue and liver give highbackground reactions.

Also, freeze/ thaw cycles have been used toimprove the penetration of probes intotissues.

PrehybridizationA prehybridization incubation is often nec-essary to prevent background staining. Theprehybridization mixture contains all com-ponents of a hybridization mixture exceptfor probe and dextran sulfate.

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Table 1: Spectral properties offluorophoresusedinFISHanalysis.Rhodamine-, fluorescein-, and coumarin-

based dyes cover the three primary colorsof the visible part of the electro-magneticspectrum. By using selective excitation andemission filters, and dichroic mirrors, thesecolors can be visualized without “cross-talk”, making triple color FISH feasible.To increase the identification of targets bycolor, a combinatorial labeling approachhas been developed (N iederlof et al., 1990;Ried et al., 1992; Wiegant et al., 1993).Multiplicity is then given by 2n – 1, wheren is the number of colors.

After establishing that hybridized probeslabeled with two haptens in different ratiosstill fluoresceat fairly constant ratios, labora-tories extended the combinatorial approachto ratio-labeling (N ederlof et al., 1992). Inratio-labeling, the ratio of fluorescenceintensities of the various color combina-tions is used for color identification of theprobes. This approach allowed simul-taneous identification of twelve differentprobes (Dauwerse et al., 1992). For detailedinformation, refer to the literaturedescribing combinatorial and ratio labeling(Wiegant and Dauwerse, 1995).

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FluorescenceUseful fluorochromes for fluorescence insitu hybridization (FISH ) include the bluefluorochrome AMCA (amino-methyl-coumarin-acetic acid), the green fluorophorefluorescein, and the red fluorochromes CY3,rhodamine, and Texas Red™. Recently, theFISH application of an infrared dye has beenreported (Ried et al., 1992), although it canonly be visualized with infrared-sensitivecameras. For routine experiments, severalimmunofluorophores with good spectralseparation properties can be used for FISHanalysis (Table 1). Chapter 5 containsdetailed procedures for using all thesefluorochromes.

Posthybridization washesLabeled probe can hybridize nonspecifi-cally to sequences which are partially butnot entirely homologous to the probesequence. Such hybrids are less stable thanperfectly matched hybrids. They can bedissociated by performing washes of variousstringencies (as described in Chapter 3). Thestringency of the washes can be manipulatedby varying the formamide concentration,salt concentration, and temperature. O ften awash in 2 x SSC containing 50% formamidesuffices. For some applications the strin-gency of the washes should be higher.H owever, we recommend hybridizingstringently rather than washing stringently.

ImmunocytochemistryIn this manual immunocytochemical proce-dures using digoxigenin, biotin, andfluorochromes are described. These allowflexibility in the choice of the final reportermolecule and type of microscopy.

Blocking reactionUse a blocking step prior to the immuno-logical procedure to remove high back-ground.

For example, perform biotin detectionsteps in PBS containing Tween® 20 andBSA (when using monoclonal antisera) ornormal serum (when using polyclonalantisera).

Perform routine digoxigenin detection inTris-H Cl buffer containing Blocking Rea-gent (instead of BSA or normal serum) toremove background. Also the addition of0.4 M N aCl can help prevent backgroundstaining if the antigen/hapten bond cansurvive the high salt conditions.

In a standard reaction, block the targettissue, cell, or chromosome preparationand then incubate it with the antibody-conjugate solution for 30 min at 37°C (or 2h at room temperature) in a moist chamber.Afterwards, wash the slide three times(5–10 min each) with buffer containingTween® 20.

Color Excitation max. (nm) Emission max. (nm)

AMCA Blue 399 446Fluorescein Green 494 523CY3 Red 552 565Rhodamine Red 555 580Texas Red Red 590 615

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When using brightfield microscopy withperoxidase, prepare the following peroxi-dase substrate solution: 0.5 mg/ml diamino-benzidine in 50 mM Tris-H Cl, pH 7.4,containing 10 mM imidazole. Shortly beforeuse, add 0.05% H 2O 2. Incubate with sub-strate from 2–30 min (depending on thesignal-to-noise ratio). We advise inspectingsections microscopically during the peroxi-dase reaction. Stop the reaction by washingthe sample with water. Counterstain withGiemsa if desired.

In the case of reflection contrast micro-scopy with peroxidase, prepare the follow-ing peroxidase substrate solution: 0.1 mg/ml diaminobenzidine in 50 mM Tris-H Cl,pH 7.4. Shortly before use, add 0.01%H 2O 2. The usual reaction time is 10 min.After stopping the reaction with H 2O anddehydrating with ethanol, evaluate slidesby oil-immersion microscopy withoutembedding.

When using Alkaline Phosphatase, preparethe following substrate: 0.16 mg/ml 5-Bromo-4-chloro-3-indolyl-phosphate (BCIP) and0.33 mg/ml N itro-blue tetrazolium salt(N BT) in 200 mM Tris-H Cl, 10 mMMgCl2, pH 9.2. Determine reaction timesby evaluating the signal-to-noise ratio,which should be checked microscopicallyduring the enzyme reaction. The reactionmedium itself is stable (in the dark). Thefinal product (N BT formazan) also hasreflective properties.

A recently introduced alkaline phospha-tase substrate (H N PP/Fast Red TR) alsoallows fluorescent detection of alkalinephosphatase label. For fluorescence micro-scopy, prepare the following substrate: 10 mg/ml H N PP (in dimethylformamide)and 25 mg/ml Fast Red TR in redist H 2O .For details on the use of H N PP/Fast RedTR, see the article in Chapter 5, page 108.

Fluorescent DN A counterstaining is usuallyperformed with red fluorescent propidiumiodide (PI) or with blue fluorescent DAPI.Currently, new counterstains are beingintroduced, e.g., green fluorescent YO YO -1(Molecular Probes, Inc., USA).

An antifading agent should be added to theembedding medium to retard fading. Whenfluorescent labels are used, the recom-mended antifading agent is Vectashield(Vector Laboratories). The different DN Acounterstains can be directly dissolved inthe embedding medium (40 ng DAPI/ml; 100 ng PI/ml; or 0.1 µM YO YO -1/ml) orthe fluorescent specimen can first becounterstained and then embedded inVectashield. If desired, the coverslip maybe sealed with nail varnish.

EnzymesUse any enzyme commonly used inimmunocytochemistry. We show examplesfor peroxidase and alkaline phosphatase.

With peroxidase (PO D), use the diamino-benzidine (DAB)/imidazole reaction (Grahamand Karnovsky, 1966). With alkaline phos-phatase (AP), use the 5-Bromo-4-chloro-3-indolylphosphate/N itro-blue tetrazolium(BCIP/N BT) reaction. The advantages ofthese colorimetric detection methods are goodlocalization properties, high sensitivity (De Jonget al., 1985; Straus, 1982; Scopsi and Larson,1986) and stability of the precipitates. Asthe DAB reaction produces a color whichcontrasts with the blue alkaline phosphatasestaining reaction (and vice versa), one canuse both enzymes in double hybridizations.Furthermore, both detection methods havebright reflective properties. They can beamplified by gold/silver (Gallyas et al.,1982; Burns et al., 1985) and the color can bemodified with heavy metal ions (H su andSoban, 1982; Cremers et al., 1987).

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al., 1985a) the DAB/peroxidase product dis-plays bright reflection when present inextremely low amounts (i.e., low localabsorption, typically A < 0.05). This propertyhas made reflection contrast microscopyinstrumental in detecting the first single copygene by nonradioactive means (Landegent etal., 1985b; H opman et al., 1986b; Ambros etal., 1986). Studies of DAB image formation inreflection contrast microscopy have shownthat best results require thin objects and lowlocal absorbances of the DAB product(Cornelese ten Velde et al., 1988).

Fluorescence microscopyA fluorescence microscope contains a lampfor excitation of the fluorescent dye and aspecial filter which transmits a high percen-tage of light emitted by the fluorescent dye.Usually antifading reagents have to be addedbefore analysis.

Fluorescence microscopy is of great valuefor nonradioactive in situ hybridization. It ishighly sensitive. Furthermore, it can be usedto excite three different immunofluoro-phores with spectrally well-separated emis-sions (which allows multiple detection).Also the option of using highly sensitiveimage acquisition systems and the relativeease of quantitation of fluorescence signalsadds to its attractiveness.

Digital imaging microscopyDigital imaging microscopes can detect signalsthat cannot be seen with conventionalmicroscopes. Also, image processing tech-nology provides enhancement of signal-to-noise ratios as well as measurement of quan-titative data. The most sensitive system todate, the cooled CCD (charged coupleddevice) camera, counts emitted photons witha high efficiency over a broad spectral range ofwavelengths, and thus is the instrument ofchoice for two-dimensional analysis. Forthree-dimensional analysis, confocal laserscanning microscopy is used.

Electron microscopyThe resolution of microscopy is enhancedwhen electrons instead of light are used,since electrons have a much shorter wave-length (0.004 nm). The practical resolvingpower of most modern electron micro-scopes is 0.1 nm. With the electron micro-scope, the fine structure of a cell can beresolved. Such analysis requires special pre-parative procedures which are described indetail in Chapter 5.

Microscopy

Brightfield microscopyIn brightfield microscopy the image isobtained by the direct transmission of lightthrough the sample.

Evaluation of in situ hybridization resultsby brightfield microscopy is preferred formost routine applications because the prep-arations are permanent. H owever, the sen-sitivity demanded by many applications (e.g.,single copy gene localization requires thedetection of a few attograms of DN A) mayrequire more sophisticated microscopy.

Darkfield microscopyIn the darkfield microscope, the illuminat-ing rays of light are directed from the side,so that only scattered light enters themicroscope lenses. Consequently, thematerial appears as an illuminated objectagainst a black background.

Darkfield microscopy is extensively used forradioactive in situ hybridization experimentsbecause large fields can be examined at lowmagnification. The distribution of the silvergrains can be seen with high contrast. In con-trast, these types of microscopy are seldomused to localize reaction products of non-radioactive hybridization procedures (H eytinget al., 1985). H owever, Garson et al. (1987)shows the application of phase contrastmicroscopy to single copy gene detection.

Phase contrast microscopyPhase contrast microscopy exploits theinterference effects produced when two setsof waves combine. This is the case whenlight passing e.g., through a relatively thickor dense part of the cell (such as the nucleus)is retarded and its phase consequentlyshifted relative to light that has passedthrough an adjacent (thinner) region of thecytoplasm. Phase contrast microscopy isoften used for enzyme detection.

Reflection contrast microscopyReflection contrast microscopy is similarto phase contrast microscopy. This tech-nique measures the shift of wavelength ofthe light reflected from the sample relativeto that of directly emitted light.

Bonnet (personal communication) madethe crucial observation that with reflectioncontrast microscopy (Ploem, 1975; Van derPloeg and Van Duijn, 1979; Landegent et

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signals. Procedures for fluorescence in situhybridization in suspension have beendescribed (Trask et al., 1985) and furtherdevelopments in this field will be veryimportant.

Flow cytometryThe enormous speed with which fluores-cence of individual cells can be measuredwith a flow cytometer has many advan-tages for quantitating in situ hybridization

Preparation of slides/coverslips– e.g., gelatin or poly-lysine treatment of slides– siliconization of coverslips

Fixation of material on slide– by precipitation (e.g., ethanol)– by cross-linkage (e.g., formaldehyde)

Pretreatment of specimen (optional)a)

b)

Treatments to prevent background staining– endogenous enzyme inactivation– RNase-treatmentPermeabilization– diluted acids– detergent/alcohol– proteases

Prehybridization (optional)Incubation of specimen with a pre-hybridization solution (= hybridization solution minus probe) is performed at the same temperature as hybridization

Denaturation of probe and target– pH or heat– simultaneous or separate denaturation of probe and target (if double stranded)

HybridizationComponents of the solution are mainly:– Denhardt’s Mix (Ficoll, BSA, PVP)– heterologous nucleic acids (e.g., herring sperm DNA/ tRNA/competitor DNA)– sodium phosphate, EDTA, SDS, salt– formamide– dextran sulfate

Post-hybridization steps– treatment with single strand specific nuclease (optional)– stringency washes

Immunological detection– blocking step– antibody incubation– colorimetric substrate or fluorescence microscopy– counterstaining– mounting

Microscopy– microscopic analysis of results and documentation

Evaluation

Probea)

b)

c)

Choice of the probe– ds: DNA, cDNA– ss: RNA, oligonucleotide ss-DNAPreparation of the probe– DNA: fragment isolation (optional)– cDNA: cloning– RNA: cloning in transcription vectors– oligonucleotides: chemical synthesisLabeling of the probe– ds DNA: random primed DNA labeling, nick translation, PCR– RNA: In vitro transcription, RT-PCR– oligonucleotides: endlabeling or tailing

Determination of hybridization conditions, e.g.,– determination of hybridization temperature, pH, use of formamide, salt concentration– composition of hybridization solution– probe concentration

Determination of hybridization specificity (controls)– nuclease treatment– use of multiple probes– use of heterologous nucleic acid/vectors

Fluorescence microscopy for probes directly labeled with a fluorochrome

Flow diagram for in situ hybridization

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Landegent, J. E., Jansen in de Wal, N.; Ommen, G. J. B.; Baas, F.; De Vijlder, J. J. M.; Van Duijn, P.;Van der Ploeg, M. (1985b) Chromosomal localizationof a unique gene by non-autoradiographic in situhybridization. Nature 317, 175–177.Landegent, J. E.; Jansen in de Wal, N.; Ploem, J. S.;Van der Ploeg, M. (1985a) Sensitive detection ofhybridocytochemical results by means of reflectioncontrast microscopy. J. Histochem. Cytochem. 33,1241–1246.Nederlof, P .M.; Van der Flier, S.; Wiegant, J.; Raap,A. K.; Tanke, H. J.; Ploem, J. S.; Van der Ploeg, M.(1990) Multiple fluorescence in situ hybridization.Cytometry 11, 126–131.Nederlof, P. M.; Van der Flier, S.; Vrolijk, J.; Tanke,H. J.; Raap, A. K. (1992) Quantification of in situhybridizationsignalsby fluorescencedigital imagingmicroscopy. III. Fluorescence ratio measurementsof double labeled probes. Cytometry 13, 838–845.Ploem, J. S. (1975) Reflection contrast microscopyas a tool for investigation of the attachment of livingcells to a glass surface. In: Van Furth, R. (Ed.)Mononuclear Phagocytes in Immunity, Infection andPathology. Oxford: Blackwell, 405–421.Reid, T.; Baldini, A.; Rand, T. C.; Ward, D. C. (1992)Simultaneousvisualization of seven different probesby in situ hybridization using combinatorialfluorescence and digital imaging microscopy. Proc.Natl. Acad. Sci. USA89, 1388–1392.Scopsi, L.; Larsson, L. I. (1986) Increased sensitivityin peroxidase immunocytochemistry. A comparativestudy of a number of peroxidase visualizationmethods employing a model system. Histochemistry84, 221–230.Straus, W. (1982) Imidazole increases the sensitivityof the cytochemical reaction for peroxidase withdiaminobenzidine at a neutral pH. J. Histochem.Cytochem. 30, 491–493.Trask, B.; Van den Engh, G.; Landegent, J.; Jansenin de Wal, N.; Van der Ploeg, M. (1985) Detection ofDNA sequences in nuclei in suspension by in situhybridization and dual beam flow cytometry.Science 230, 1401–1403.Van der Ploeg, M.; Van Duijn, P. (1979) Reflectionversus fluorescence. Histochemistry 62, 227–232.

Wiegant, J.; Wiesmeijer, C. C.; Hoovers, J. M. N.;Schuuring, E.; D’Azzo, A.; Vrolijk, J.; Tanke, H. J.;Raap, A. K. (1993) Multiple and sensitive in situhybridization with rhodamine-, fluorescein-, andcoumarin-labeled DNAs. Cytogenet. Cell Genet. 63,73–76.Wiegant, J.; Dauwerse, J. G. (1995) Multiple-coloredchromosomes by fluorescence in situ hybridization.In: Verma, R. S.; Babu, A. (Eds.) Human Chromo-somes: Principles and Techniques, 2nd ed. New York: McGraw-Hill, Inc.

ReferencesAmbros, P. F.; Matzke, M. A.; Matzke, A. J. M.(1986) Detection of a 17 kb unique (T-DNA) in plantchromosomes by in situ hybridization. Chromosoma94, 11–16.Burns, J.; Chan, V. T. W.; Jonasson, J. A.; Fleming, K. A.; Taylor, S.; McGee, J. O. D. (1985) Sensitivesystem for visualizing biotinylated DNA probeshybridized in situ: rapid sex determination of intactcells. J. Clin. Pathol. 38, 1085–1092.Cornelese ten Velde, I.; Bonnet, J.; Tanke, H. J.;Ploem, J. S. (1988) Reflection contrast microscopy.Visualization of (peroxidase generated) diamino-benzidine polymer products and its underlyingoptical phenomena. Histochemistry 89, 141–150.Cremers, A. F. M.; Jansen in de Wal, N.; Wiegant, J.;Dirks, R. W.; Weisbeek, P.; Van der Ploeg, M.;Landegent, J. E. (1987) Nonradioactive in situhybridization. A comparison of several immuno-cytochemical detection systems using reflectioncontrast microscopy and electron microscopy. Histochemistry 86, 609–615.Dauwerse, J. G.; Wiegant, J.; Raap, A. K.; Breuning,M. H.; Van Ommen, G. J. B. (1992) Multiple colorsby fluorescence in situ hybridization using ratio-labeled DNA probes create a molecular karyotype.Hum. Molec. Genet. 1, 593–598.De Jong, A. S. H.; Van Kessel-Van Vark, M.; Raap, A.K. (1985)Sensitivityof variousvisualizationmethodsfor peroxidase and alkaline phosphatase activity inimmunoenzyme histochemistry. Histochem. J. 17,1119–1130.Gallyas, F.; Gorcs, T.; Merchenthaler, I. (1982) High grade intensification of the end-product of the diaminobenzidine reaction for peroxidase histo-chemistry. J. Histochem. Cytochem. 30, 183–184.Garson, J. A.; Van den Berghe, J. A.; Kemshead, J. T. (1987) Novel non-isotopic in situ hybridizationtechnique detects small (1 kb) unique sequences in routinely G-banded human chromosomes: finemapping of N-myc and b-NGF genes. Nucleic AcidsRes. 15, 4761–4770.Graham, R. C.; Karnovsky, M. J. (1966) The earlystages of absorption of injected horseradish peroxi-dase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291–302.Heyting, C.; Kroes, W. G. M.; Kriek, E.; Meyer, I.;Slater, R. (1985) Hybridization of N-acetoxy-N-acetyl-2-aminofluorene labeled RNA to Q-banded meta-phase chromosomes. Acta Histochem. 77, 177–184.

Hopman, A. H. N.; Wiegant, J.; Raap, A. K.;Landegent, J. E.; Van der Ploeg, M.; Van Duijn, P.(1986b) Bi-color detection of two target DNAs bynonradioactive in situ hybridization. Histochemistry85, 1–4.Hsu, S. M.; Soban, E. (1982) Color modification ofdiaminobenzidine (DAB) precipitation by metallicions and its application for double immunohisto-chemistry. J. Histochem. Cytochem. 30, 1079–1082.

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Procedures for In Situ Hybridization

to Chromosomes Cells, and Tissue Sections

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This chapter includes contributions fromseveral leading scientists, from researchersin molecular biology to clinical pathol-ogists, who practice in situ hybridization.Protocols are given for in situ hybridiza-tions on widely varying substrates, e.g.,chromosome spreads, chromosomes insuspension, single cells, paraffin-embeddedtissue sections, ultrathin tissue sections, andwhole mount preparations. H ybridizationmethods are described for both DN A andRN A targets.

Applications covered include gene mapping,gene expression, developmental biology,tumor biology, cell sorting, clinical cyto-genetics, and analysis of infectious diseases.

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These procedures use digoxigenin, biotin,and fluorochromes for labeling DN A,RN A and oligonucleotides. Labeling tech-niques include the classical methods, such asrandom primed DN A labeling, nick trans-lation, and oligonucleotide tailing withterminal transferase, as well as more modernmethods such as PCR and “PRIN S”, anovel and highly sensitive approach to insitu hybridization, in which the probe islabeled after its hybridization to the targetnucleic acid.

Please note that the protocols have beenoptimized by members of the individuallaboratories and can be varied if necessary.

Procedures for In SituHybridization toChromosomes, Cells, and Tissue Sections

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Procedures for In Situ H ybridization to Chromosomes, Cells, and Tissue Sections

Contents

ISH to whole chromosomesIn situ hybridization to human metaphase chromosomes using DIG-, biotin- or fluorochrome-labeled DNA probes and detection with fluorochrome conjugates. 62J. Wiegant, Department of Cytochemistry and Cytometry, University of Leiden, TheN etherlands; U . Mathieu and P. L ichter, German Cancer Research Center,H eidelberg, Germany; J. Wienberg and N . Arnold, Institute for Anthropology andH uman Genetics, University of Munich, Germany; S. Popp and T. Cremer, Institutefor H uman Genetics and Anthropology, University of H eidelberg, Germany; D . C .Ward, H uman Genetics Department, Yale University, N ew H aven, Connecticut,USA ; S. Joos and P. L ichter, German Cancer Research Center, H eidelberg, Germany.

Detection of chromosomal imbalances using DOP-PCR and comparative genomic hybridization (CGH). 72S. Joos, B. Schütz , M. Bentz , and P. L ichter, German Cancer Research Center,H eidelberg, Germany.

Identification of chromosomes in metaphase spreads and interphase nuclei with PRINS Oligonucleotide Primers and DIG- or rhodamine-labeled nucleotides. 79R. Seibl, Research Laboratories, Boehringer Mannheim GmbH , Penzberg, Germany.

Identification of chromosomes in metaphase spreads with DIG- or fluorescein-labeled human chromosome-specific satellite DNA probes. 88G. Sagner, Research Laboratories, Boehringer Mannheim GmbH , Penzberg, Germany.

Fluorescence in situ hybridization of a repetitive DNA probe to human chromosomes in suspension. 94D. Celeda, U . Bettag, and C . Cremer, Institute for Applied Physics and Institute forH uman Genetics and Anthropology, University of H eidelberg, Germany.

A simplified and efficient protocol for nonradioactive in situ hybridization to polytene chromosomes with a DIG-labeled DNA probe. 97E. R . Schmidt, Institute for Genetics, Johannes Gutenberg-University of Mainz , Germany.

Multiple-target DNA in situ hybridization with enzyme-based cytochemical detection systems. 100E. J. M. Speel, F. C . S. Rameaekers, and A . H . N . H opman, Department of MolecularCell Biology & Genetics, University of L imburg, Maastricht, The N etherlands.

DNA in situ hybridization with an alkaline phosphatase-based fluorescent detection system. 108G. Sagner, Research Laboratories, Boehringer Mannheim GmbH , Penzberg, Germany.

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ISH to cellsCombined DNA in situ hybridization and immunocytochemistry for the simultaneous detection of nucleic acid sequences, proteins, and incorporated BrdU in cell preparations. 110E. J. M. Speel, F. C . S. Rameaekers, and A . H . N . H opman, Department of MolecularCell Biology & Genetics, University of L imburg, Maastricht, The N etherlands.

In situ hybridization to mRNA in in vitro cultured cells with DNA probes. 116Department of Cytochemistry and Cytometry, University of Leiden, TheN etherlands.

Identification of single bacterial cells using DIG-labeled oligonucleotides. 119B. Zarda, R . Amann, and K.-H . Schleifer, Institute for Microbiology, Technical University of Munich, Germany.

ISH to tissuesDetection of HPV11 DNA in paraffin-embedded laryngeal tissue with a DIG-labeled DNA probe. 122J. Rolighed and H . L indeberg, EN T-department and Institute for Pathology AarhusUniversity H ospital, Denmark .

Detection of mRNA in tissue sections using DIG-labeled RNA and oligonucleotide probes. 126P. Komminoth, D iv ision of Cell and Molecular Pathology, Department of Pathology,University of Zürich, Switzerland.

Detection of mRNA on paraffin embedded material of the central nervous system with DIG-labeled RNA probes. 136H . Breitschopf and G . Suchanek , Research Unit for Experimental N europathology,Austrian Academy of Sciences, V ienna, Austria.

RNA-RNA in situ hybridization using DIG-labeled probes: the effect of high molecular weight polyvinyl alcohol on the alkaline phosphatase indoxyl-nitroblue tetrazolium reaction. 141M. De Block and D . Debrouwer, Plant Genetic Systems N .V., Gent, Belgium.

Detection of neuropeptide mRNAs in tissue sections using oligonucleotides tailed with fluorescein-12-dUTP or DIG-dUTP. 146Department of Cytochemistry and Cytometry, University of Leiden, TheN etherlands.

In situ hybridization of DIG-labeled rRNA probes to mouse liver ultrathin sections. 148D. Fischer, D . Weisenberger, and U . Scheer, Institute for Zoology I , University ofWürzburg, Germany.

RNA in situ hybridization using DIG-labeled cRNA probes. 152H . B. P. M. Dijkman, S. Mentzel, A . S. de Jong, and K. J. M. Assmann, Department ofPathology, N ijmegen University H ospital, N ijmegen, The N etherlands.

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Whole mount ISHLocalization of the expression of the segmentation gene hunchback in Drosophila embryos with DIG-labeled DNA probes. 158D. Tautz , Institute for Genetics and Microbiology, University of Munich, Germany.

Detection of even-skipped transcripts in Drosophila embryos with PCR/DIG-labeled DNA probes. 162N . Patel and C . Goodman, Carnegie Institute of Washington, EmbryologyDepartment, Baltimore, Maryland, USA.

Whole mount fluorescence in situ hybridization (FISH) of repetitive DNA sequences on interphase nuclei of the small cruciferous plant Arabidopsis thaliana. 165Serge Bauwens and Patrick Van O ostveldt, Laboratory for Genetics and Laboratoryfor Biochemistry and Molecular Cytology, Gent University, Gent, Belgium.

II. Denaturation and hybridizationFor denaturation and hybridization threealternative protocols are given:A. For probes recognizing repetitive

targets such as the alphoid sequencesB. For probes recognizing unique

sequencesC. For probes or probe cocktails which

contain repetitive sequences occurringthroughout the genome (e.g., Alu sequences) [competition hybridization]

A. For repetitive probes!1 Using digoxigenin- (DIG-), biotin-, or

any fluorochrome-labeled nucleotide,label 1 µg of DN A probe according tothe procedures described in Chapter 4of this manual.

!2 Precipitate the labeled probe withethanol.

!3 Prepare a probe stock solution as follows:" Resuspend the precipitated probe at a

concentration of 10 ng/µl in a solu-tion containing 60% deionized form-amide, 2 x SSC, 50 mM sodiumphosphate; pH 7.0.

" Incubate the tube for 15 min at 37°Cwith occasional vortexing until theprecipitated DN A dissolves.

!4 Dilute the probe from the stock to thedesired concentration (typically 2 ng/µl)in a solution containing 60% deionizedformamide, 2 x SSC, 50 mM sodiumphosphate; pH 7.0.

!5 Apply 5 µl diluted probe under a cover-slip on the object slide and place theslide in an oven at 80°C for 2–4 min todenature the probe and target. Note: Sealing with rubber cement is notmandatory.

!6 To hybridize, place the slides in a moistchamber at 37°C overnight.

!7 Wash the slide as follows:" 3 x 5 min at 37°C with 2 x SSC con-

taining 60% formamide." 1 x 5 min with the buffer to be used

for immunological detection.

In recent years, a number of improvementsin chromosomal in situ hybridization pro-tocols have been achieved. These haveallowed a fairly high success rate for singlecopy gene localization as well as competi-tion in situ hybridization. H ere we presenta detailed outline of the procedure that hasbeen successfully applied in variouslaboratories.

The procedures have been optimized forfluorescent detection. For the second editionof this manual, we have included newprocedures and new illustrations for multi-color, multitarget fluorescent detection.

The procedures have also been used withslight modifications by the groups of Dr. P.Lichter, Dr. J. Wienberg, Dr. T. Cremerand Dr. D. Ward. The illustrative materialthey have provided below demonstratesthe flexibility and wide applicability of thetechnique.

At the end of the article, Dr. Lichter’sgroup has provided a troubleshootingguide to help solve hybridization problemsthat may occur.

I. Pretreatment of metaphase spreads on slides (optional)!1 Incubate metaphase spreads with 100 µl

of 100 µg/ml RN ase A (in 2 x SSC)under a coverslip for 1 h at 37°C.

!2 Wash slides 3 x 5 min with 2 x SSC.!3 Dehydrate slides in an ethanol series

(increasing ethanol concentrations).!4 Incubate with 0.005–0.02% pepsin in

10 mM H Cl for 10 min at 37°C.!5 Wash slides as follows:

" 2 x 5 min with PBS." O nce with PBS containing 50 mM

MgCl2.!6 Post-fix for 10 min at room temperature

with a solution of PBScontaining 50 mMMgCl2 and 1% formaldehyde.

!7 Wash with PBS and dehydrate (as inStep 3 above).

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Procedures for In Situ H ybridization to Chromosomes, Cells, and Tissue SectionsISH to whole chromosomes

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In situhybridization to human metaphase chromosomes using DIG-, biotin-, or fluorochrome-labeled DNA probes and detection with fluorochromeconjugatesJ. Wiegant, Department of Cytochemistry and Cytometry, Leiden University, N etherlands.

!3 Prepare a probe stock solution bydissolving the pellet in a solution of50% deionized formamide, 2 x SSC,10% dextran sulfate, 50 mM sodiumphosphate; pH 7. Depending on theprobe cocktail, the final concentrationranges from 10–40 ng/µl. Mix well!Examples: Prepare a 10 ng/µl stocksolution of cosmid probes, a 20 ng/ µlstock of chromosome specific libraries,or a 40 ng/µl stock of YAC probes.

!4 For hybridization, dilute the probe fromthe stock to the desired concentration ina solution of 50% deionized formamide,2 x SSC, 10% dextran sulfate, 50 mMsodium phosphate; pH 7.Examples: For hybridization, use2–5 ng/µl of cosmid probes, 10–20 ng/µlof chromosome specific libraries, or20–40 ng/µl of YAC probes.

!5 Denature the probe at 75°C for 5 min,chill on ice, and allow annealing ofthe repetitive elements at 37°C for either 30 min (if C ot-1 DN A is used ascompetitor) or 2 h (if total humanplacenta DN A is used as competitor).

!6 Prepare the chromosomes on the slideby performing the following steps:" To the chromosomes, add a solution

of 70% formamide, 2 x SSC, 50 mMsodium phosphate; pH 7. Cover witha coverslip.

" Incubate in an oven at 80°C for 2–4min to denature the chromosomes.

" Remove the coverslip and quench thechromosomes in chilled 70% ethanol.

" Dehydrate the slide by passing it through 90% ethanol, then 100% alcohol.

" Air dry the slide, preferably on ametal plate at 37°C.

!7 H ybridize the chromosomes overnightwith 10 µl of the pre-annealed probe(from Step 5) under a sealed coverslip.


taining 50% formamide." 3 x 5 min at 60°C with 0.1 x SSC." 1 x 5 min with the buffer to be used in

immunological detection.

B. For unique probes!1 Prepare the probe stock solution as

described under Procedure IIA, but usea solution containing 50% deionizedformamide, 2 x SSC, 10% dextran sul-fate, 50 mM sodium phosphate; pH 7.

!2 Dilute 2–5 µl of the probe stock solu-tion to 10 µl with a solution containing50% deionized formamide, 2 x SSC,10% dextran sulfate, 50 mM sodiumphosphate; pH 7. Mix well!

!3 To denature probe and target, do thefollowing:" Apply 10 µl of the dilute hybridiza-

tion solution under a coverslip on theobject slide.

" Seal coverslip to slide with rubbercement.

" Place the slide in an oven at 80°C for2–4 min.

!4 To hybridize, place the slides in a moistchamber at 37°C overnight.


taining 50% formamide." 5 x 2 min with 2 x SSC." 1 x 5 min with the buffer used in

detection.

C. For probescontainingrepetitiveelements [competitive hybridization]!1 Using DIG-, biotin-, or any fluoro-

chrome-labeled nucleotides, label 1 µgof DN A probe according to theprocedures described in Chapter 4 ofthis manual.

!2 To precipitate the labeled probe, do thefollowing:" Add a 50-fold excess of human C ot-1

DN A (or 500-fold excess fragmentedhuman placenta DN A), a 50-foldexcess of fragmented herring spermDN A, a 50-fold excess of yeasttRN A, 1/10th volume of 3 M sodiumacetate ( pH 5.6), and 2.5 volumes ofcold (–20°C) ethanol.

" Incubate for 30 min on ice." Centrifuge for 30 min at 4°C." Discard the supernatant and dry the

pellet.

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B1. Digoxigenin-labeled probe, low sensitivity!1 Wash slides briefly with TN T buffer

[100 mM Tris-H Cl (pH 7.5), 150 mMN aCl, 0.05% Tween 20].

!2 Incubate for 30 min at 37°C with TN Bbuffer [100 mM Tris-H Cl (pH 7.5), 150 mM N aCl, 0.5% blocking reagent].

!3 Incubate for 30 min at 37°C with theproper dilution (typically 2 µg/ml, inTN B) of a fluorescently labeled sheepanti-DIG antibody.

!4 Wash the slides (3 x 5 min) with TN T.!5 Prepare the slides for viewing as in the

biotin low sensitivity procedure (Pro-cedure IIIA1), Step 5.

B2. Digoxigenin-labeled probe, high sensitivity!1 Wash slides briefly with TN T buffer

[100 mM Tris-H Cl (pH 7.5), 150 mMN aCl, 0.05% Tween® 20].

!2 Pipette 100 µl of TN B buffer [100 mMTris-H Cl (pH 7.5), 150 mM N aCl,0.5% blocking reagent] onto each slideand cover with a 24 x 50 mm coverslip.Incubate for 30 min at 37°C in a moistchamber (1 L beaker containing mois-tened tissues, covered with aluminumfoil).

!3 Immerse the slides for 5 min in TN T toloosen the coverslips.

!4 Prepare fresh working solutions of threeantibodies:" Antibody 1 working solution: mouse

monoclonal anti-DIG, 0.5 µg/ml inTN B.

" Antibody 2 working solution: DIG-conjugated sheep anti-mouse Ig,2 µg/ml in TN B.

" Antibody 3 working solution: fluores-cein- or rhodamine-conjugated sheepanti-DIG, 2 µg/ml in TN B.Note: The three antibodies used inthis procedure are also available in theFluorescent Antibody Enhancer Setfor DIG Detection.

!5 Pipette 100 µl of antibody 1 working solu-tion onto each slide and cover with a24 x50mm coverslip.Incubatefor 30min at37°C in a moist chamber.

III. Single color fluorescent detectionwith immunological amplificationA1. Biotin-labeled probe, low sensitivity!1 Wash slides briefly with TN T buffer



!3 Incubate for 30 min at 37°C with theproper dilution (typically 10 µg/ml) of afluorescently labeled streptavidin conju-gate in TN B.

!4 Wash the slides (3 x 5 min) with TN T.!5 Prepare the slides for viewing by per-

forming the following steps:" Dehydrate through an ethanol series

(70% , then 90% , then 100% ethanol;5 min each).

" Air dry." Stain with the appropriate DN A

counterstain." Embed in Vectashield (Vector).

!6 View the slides by fluorescence micro-scopy, using appropriate filters.

A2. Biotin-labeled probe, high sensitivity!1 Wash slides briefly with TN T buffer



!3 Incubate for 30 min at 37°C with theproper dilution (typically 10 µg/ml) of afluorescently labeled streptavidin conju-gate in TN B.

!4 Wash the slides (3 x 5 min) with TN T.!5 Incubate the slides for 30 min at 37°C

with the proper dilution of biotinylatedgoat anti-streptavidin (5 µg/ml) in TN B.

!6 Repeat the washes as in Step 4.!7 Repeat Step 3.!8 Repeat the washes as in Step 4.!9 Prepare the slides for viewing as in the


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IV. Multicolor fluorescence in situhybridization (Multicolor FISH)For multicolor FISH , several sets of probeswith different labels must be hybridizedsimultaneously to the target and visualizedwith combinations of antibodies.

Table 1 lists an antibody-probe matrixwhich allows a triple color detection ofthree differently labeled chromosomespecific libraries (so-called chromosomepainting probes).

!6 Wash the slides (3 x 5 min) at roomtemperature with TN T.

!7 Pipette 100 µl of antibody 2 workingsolution onto each slide and add a 24 x50 mm coverslip. Incubate for 30 min at37°C in a moist chamber.

!8 Repeat the washes as in Step 6.!9 Pipette 100 µl of antibody 3 working

solution onto each slide and add a 24 x50 mm coverslip. Incubate for 30 min at37°C in a moist chamber.Note: For even higher sensitiv ity, repeatSteps 5–8 before performing the incu-bation with antibody 3 (Step 9).

!10 Repeat the washes as in Step 6.!11 Prepare the slides for viewing as in the


C1. Fluorescently labeled probes (fluorescein, coumarin, CY3, rhodamine,Texas Red™), low sensitivityAfter the posthybridization washes (Pro-cedure II), prepare the slides for viewing asin the biotin low sensitivity procedure(Procedure IIIA1), Step 5.

C2. Fluorescein-labeled probes, high sensitivity!1 Wash slides briefly with TN T buffer



!3 Incubate for 30 min at 37°C with theproper dilution of an anti-fluoresceinantibody (either polyclonal or mono-clonal) in TN B.

!4 Wash the slides (3 x 5 min) with TN T.!5 Depending on the antibody used in Step 3,

incubate with the proper dilution of afluorescently labeled rabbit anti-mouseor goat anti-rabbit antibody in TN B for30 min at 37°C.

!6 Repeat the washes as in Step 4.!7 Prepare the slides for viewing as in the

biotin low sensitivity procedure (Proce-dure IIIA1), Step 5.

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Incubation Probe 1 Probe 2 Probe 3Biotin Digoxigenin Fluorescein

1 Streptavidin- Mouse anti- Rabbit anti-Texas Red™ DIG antibody fluorescein antibody

2 Biotin-labeled Sheep anti-mouse FITC-labeled goatanti-streptavidin antibody anti-rabbit antibody

3 Streptavidin- AMCA-labeledTexas Red™ sheep anti-DIG

antibody

Note:Be careful that the selected antibodiesdo not cross-react.

Outline of protocol for multicolor FISH!1 Simultaneously hybridize all probes

listed in Table 1 to the target.!2 For each of the 3 detection incubations,

perform the following steps:" Prepare the antibodies needed in the

incubation (as listed in Table 1) at theproper dilution in the correct buffer.Note: Use the procedures in SectionI I I above as a guideline.

" Mix all antibodies needed in theincubation and incubate simultane-ously with the slide.

" Perform blocking and washing stepsas in Section III above.

!3 Repeat Step 2 until all incubations arecomplete.

!4 Do not counterstain the slides, butdehydrate, air dry, and mount the slidesas in the biotin low sensitivity proce-dure (Procedure IIIA1), Step 5.

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B. Detection of a trisomy in a leukemia patient in interphase nuclei using doublein situ hybridizationfrom U. Mathieu and Dr. P. L ichter,German Cancer Research Center,H eidelberg, Germany.

N ick translation was used to label achromosome 17-specific alphoid DN A (withdigoxigenin) and a chromosome 18-specificalphoid DN A (with biotin). Both the dig-oxigenin-labeled and the biotin-labeledprobes were hybridized to a chromosomesample from a leukemia patient. The pro-cedures described above for repetitive DN A(Procedure IIA of this article) were used forslide preparation, labeling, and hybridi-zation, except the following minor modi-fications were necessary for alphoid DN A:!1 After nick translation labeling, concen-

trate the probe (approx. 10–30 ng DN A)by drying the DN A directly in the tube.Note: Ethanol precipitation is notnecessary.

!2 Dissolve the dried DN A pellet in 60%formamide, 2 x SSC. Note: Do not use dextran sulfate in theresuspension buffer. O mission ofdextran sulfate reduces the chance ofcross-hybridization.

!3 After hybridization, use the followingwashes:" 3 x 5 min at 42°C with 60% forma-

mide, 2 x SSC; pH 7.Note: Compared to the standardprotocol the concentration of forma-mide is increased up to 60% .

Figure1: FISH of CY3-labeledsatelliteIII probepUC1.77(chromo-some 1) to human lymphocytemetaphase and interphase cells.The slides were counterstained withYOYO-1. The photomicrograph wastaken with a double band-passfluorescence filter to allow simul-taneous visualization of fluoresceinand Texas Red™.

V. Results obtained with human metaphase chromosome spreadsThe following examples (from ourlaboratory and other laboratories) showdifferent applications of the proceduresdescribed in this section .

A. Multicolor FISH of human lymphocytemetaphase and interphase cellsfrom the Department of Cytochemistryand Cytometry, Leiden University, N eth-erlands

The techniques described above allow thedetection of two, three, or many differentprobes simultaneously (Figures 1–4).

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Figure 4: Twelve color FISH of 12 different ratio-labeled chromosome-specific libraries to humanlymphocyte metaphase and interphase cells.For details of the experiment, see Dauwerse et al.(1992). The photomicrograph was created by super-imposing the coumarin image and the image ob-tained with a double band-pass fluorescence filter(to allow simultaneous visualizationof fluoresceinandTexasRed™).

Figure 2: Triple color FISH of coumarin-labeled satellite IIIprobe pUC1.77 (chromosome 1),fluorescein-labeled alphoid probepBamX5 (chromosome X), andrhodamine-labeled alphoid probep17H8 (chromosome 17) to humanlymphocyte metaphase andinterphase cells. Thephotomicrograph was taken bysuperimposing the coumarin,fluorescein, and rhodamine images.

"

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Figure 3: Double color FISH of a biotin-labeledchromosome1-specificlibraryandadigoxigenin-labeled chromosome 4-specific library to humanlymphocyte metaphase and interphase cells.The libraries were visualized by a combinedimmunocytochemical reaction using fluorescein-labeled anti-digoxigenin and Texas Red™-labeledstreptavidin. Thephotomicrographwas taken with adouble band-pass fluorescence filter to allowsimultaneous visualization of fluorescein and Texas

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Figure 5: Double color FISH of a digoxigenin-labeled chromosome 17-specific probe and abiotin-labeled chromosome 18-specific probe to a human chromosome preparation containinga trisomy. Each alphoid DNA probe was specific for the centromere of its target chromosome. In themetaphase and in the interphase, the probes de-tected three signals (green) from chromosome 18and two signals (red) from chromosome 17. DAPIwas used for counterstaining.

Figure 5 shows the result of the hybridi-zation. The digoxigenin-labeled probe wasvisualized with anti-DIG-rhodamine; thebiotin-labeled probe, with avidin-FITC.

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Figure 6: Double color FISH of a digoxigenin-labeled chromosome 14-specific cosmid probeand a biotin-labeled chromosome 21-specificcosmid probe to a human chromosome prepa-rationof a leukemia patient containing a trisomyand a tetrasomy. In the interphase nuclei, thecosmid DNA probes detected three copies (red) ofchromosome 14 and four copies (green) ofchromosome 21. DAPI was used for

Figure 7: Double color FISH todetect the Philadelphiachromosome (Ph1) in a patientwith ALL. A cosmid probe specificfor chromosome 9 and a YAC probespecific for chromosome 22 werehybridized to interphase nuclei(Bentz et al., 1994). These twoprobes detected two red(rhodamine) signals fromchromosomes 22, one green (FITC)signal from chromosome 9 and oneyellow signal caused by theoverlapping of red (chromosome 22)and green (chromosome 9) signalson Ph1. DAPI was used for counter-staining.

Reprinted from Bentz et al. (1994)Detection of Chimeric BCR–ABL genes onBone Marrow Samples and Blood Smearsin Chronic Myeloid and AcuteLymphoblastic Leukemia by In SituHybridization,Blood 83 (7); 1922–1928 with permissionof the Journal Permissions Department,W. B. Saunder’s Company.

Figure8:FISHof a chromosome2-specific libraryto chromosomes of Gorilla gorilla. StandardchromosomepreparationswereobtainedfromEBV-transformed lymphoblastoidcell lines. DNAprobeswere from flow-sorted human chromosomes clonedto phages or plasmids. Probes were labeled withdigoxigeninby nick translation. Detection was per-formed with monoclonal mouse anti-DIG antibody,rabbit anti-mouse-TRITC, andgoat anti-rabbit-TRITC.

C. Detection of a trisomy and a tetrasomyin interphase nuclei of a leukemia patientwith two different cosmid probesfrom U. Mathieu and Dr. P. L ichter

A digoxigenin-labeled cosmid probe specif-ic for chromosome 14 and a biotin-labeledcosmid probe specific for chromosome 21were used to detect polyploidy in aleukemia patient (Figure 6). Probes werelabeled by nick translation and visualizedwith either anti-DIG-rhodamine (chromo-some 14-specific probe) or avidin-FITC(chromosome 21-specific probe).

D. Interphase nuclei of a patient with Ph1-positiveALL(acute lymphoblasticleukemia)from M. Bentz , P. L ichter, H . Döhner andG. Cabot.

The Philadelphia chromosome (Ph1) is thederivative of a translocation betweenchromosome 9 and chromosome 22 [(t9;22)(q34;q11)]. Figure 7 shows the use of twoprobes, one specific for chromosome 9 andthe other specific for chromosome 22, todetect Ph1.

E. Chromosomal in situ suppression (CISS)hybridization of the human DNA libraryspecific for chromosome 2 to chromo-somes of Gorilla gorillafrom Dr. J. Wienberg and Dr. N . Arnold,Institute for Anthropology and H umanGenetics, University of Munich, Germany.

The hybridization pattern (Figure 8) ob-tained with specific DN A probe sets(Weinberg et al., 1990) confirms theassumed fusion of two submetacentricchromosomes during human evolution.

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Figure 9: Two-color chromosomal insitusuppression(CISS)-hybridization of a metaphase spreadobtained from a patient with chronic myeloid leukemia 46, XY, t(9;22) (q34;q11). In Panel a, the probe was a digoxigenin-labeled chromosome 22-specific library, which was detected with a mouse anti-DIG antibody and a FITC-conjugated sheep anti-mouse antibody. The same metaphase spread wassimultaneously painted (Panel b) with a biotin-labeled chromosome 9-specific library, which was detectedwith avidin conjugated to the fluorochrome AMCA (aminocoumarin). Chromosomes were counterstained withpropidium iodide. Chromosomal analysis using GTG-banded chromosomes was kindly performed by R. Becher, Westdeutsches Tumorzentrum, Essen.

Figure 10: FISH of an ATPase-specific probe and a probecontaining L1-repetitivesequences to female mousechromosomes. Yellow color(banding) isdue to the biotin-labeledprobe containing L1-repetitivesequences, which was detectedwith FITC. Red „dots“ indicate asignal from each of the four (lookclosely) chromatids obtained withthe digoxigenin-labeled ATPase-specific probe, which was detectedwith anti-DIG Fab fragments (fromsheep) and Texas Red™-conjugatedanti-sheep Ig Fab. The blue color isthe DAPI counterstain.

clearly painted the normal chromosome 22(bottom), the Philadelphia chromosome(triangle) and the translocated material22q11 → 22qter contained in the derivativechromosome 9 (arrow). In Panel b, thechromosome 9-specific probe has stainedthe normal chromosome 9 blue except forthe centromeric region, while in the deriv-ative chromosome 9, the region 9pter →9q34 is also painted. The arrow points tothe breakpoint on chromosome 9.

F. Illustration of a chromosomaltranslocation from a patient with chronic myeloid leukemiafrom S. Popp and Dr. T. Cremer, Institutefor H uman Genetics and Anthropology,University of H eidelberg, Germany.

The chromosomal translocation was de-tected by using library probes from sortedchromosomes 9 and 22 (Figure 9). In Panel a,the chromosome 22-specific probe has

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G. Digitized images of female mousechromosomesvisualizedwithaZeissepifluorescent microscope equipped with acooled CCD camerafrom Dr. D . C . Ward, H uman GeneticsDepartment, Yale University, N ewH aven, CT, USA.

In Figure 10, a probe specific for N a+/K+

ATPase alpha subunits and a probe contain-ing an L1-repetitive sequence were hybrid-ized to female mouse chromosomes.

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Reagents available from Boehringer Mannheim for these procedures

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Reagent Description Cat. No. Pack size DIG-Nick Translation 5x conc. stabilized reaction buffer in 50% 1745 816 160 µl Mix* for in situ probes glycerol (v/v)andDNAPolymeraseI, DNaseI, (40 labeling

0.25 mMdATP, 0.25 mMdCTP, 0.25 mMdGTP, reactions)0.17 mM dTTP and 0.08 mM DIG-11-dUTP.

Biotin-Nick Translation 5x conc. stabilized reaction buffer in 50% 1745 824 160 µl Mix* for in situ probes glycerol (v/v)andDNAPolymeraseI, DNase I, (40 labeling

0.25 mM dATP, 0.25 mM dCTP, 0.25 mM dGTP, reactions)0.17 mM dTTP and 0.08 mM biotin-16-dUTP.

Nick Translation Mix* 5x conc. stabilized reaction buffer in 50% 1745 808 200 µl for in situ probes glycerol, DNA Polymerase I and DNase I.dNTP Set Set of dATP, dCTP, dGTP, dTTP, 1277 049 4x10 µmol

100 mM solutions, lithium salts. (100 µl)Fluorescein-12-dUTP Tetralithium salt, 1 mM solution 1373 242 25 nmol

(25 µl)Tetramethyl- Tetralithium salt, 1 mM solution 1534 378 25 nmolrhodamine-6-dUTP (25 µl)AMCA-6-dUTP Tetralithium salt, 1 mM solution 1534 386 25 nmol

(25 µl)RNase A Pyrimidine specific endoribonuclease 109 142 25 mg

which acts on single-stranded RNA. 109 169 100 mgPepsin Aspartic endopeptidase with broad 108 057 1 g

specificity. 1693 387 5 gDNA, COT-1, human COT-1 DNA is used in chromosomal in situ 1581 074 500 µg

suppression [CISS] hybridization. (500 µl)DNA, MB-grade The ready-to-use solution is directly 1467140 500 mg from fish sperm added to the hybridization mix. (50 ml)tRNA Lyophilizate, 1 mg of dry substance 109 495 100 mgfrom baker’s yeast corresponds to 16 A260 units. 109 509 500 mgTween® 20 Aqueous solution, 10% (w/v) 1332 465 5x10 mlTris Powder 127 434 100 g

708 968 500 g708 976 1 kg

Blocking Reagent, Powder 1096 176 50 g for nucleic acid hybridizationAnti-Digoxigenin, For the detection of digoxigenin-labeled 1333 062 100 µg clone 1.71.256, compoundsmouse IgG1, !Anti-Digoxigenin- For the detection of digoxigenin-labeled 1207741 200 µg Fluorescein, Fab compoundsfragments from sheepAnti-Digoxigenin- For the detection of digoxigenin-labeled 1207750 200 µg Rhodamine, Fab compoundsfragments from sheepAnti-Mouse Ig-Digoxi- Antibody for triple color FISH 1214 624 200 µg genin, F(ab)2 fragment Streptavidin-AMCA For the detection of biotin-labeled compounds. 1428 578 1 mgStreptavidin-Fluorescein For the detection of biotin-labeled compounds. 1055 097 1 mgDAPI Fluorescence dye for staining of 236 276 10 mg 4',6-Diamidine-2'-Phenyl- chromosomesindole Dihydrochloride

*This product or the use of thisproduct may be covered by one or more patents of BoehringerMannheim GmbH, including thefollowing: EP patent 0 649 909(application pending).

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" I f the probe is larger than 500 nt, add more DN ase I (roughly about 1–10 ng) to the probe sample kept onice, then incubate longer at 15°C. Note: Usually higher concentrationsof DN ase must be added for anadditional 30 min incubation. A fterthe incubation, analyze the probe ona gel as above.

" I f the DN A is almost or completelyundigested, purify the probe and repeat the labeling step.I f part of the DN A is smaller than100 nt, repeat the labeling step usingless DN ase I.I f all the DN A is smaller than 100 nt,repurify the probe (to remove anypossible contaminating DN ase) andrepeat the labeling step.

!4 Check whether the fluorochromes haveseparated from the detecting moleculesby passing the solution of fluorochrome-conjugated molecules through a Q uickSpin™ column (G-50). Then determinethe state of the molecules:" I f color remains in flow through,

fluorochromes are still conjugated." If color remains in the upper part

of the column, fluorochromes haveseparated from the detecting molecules.

!5 Modify chromosome denaturationprocedure (Procedure II in this article)by either varying the time and temper-ature of the denaturation step or byfurther pepsin digestion according to thefollowing protocol:Note: Digestion w ith pepsin cansignificantly improve probe penetra-tion, but overdigestion can also occur.Pepsin treatment is often useful whenspecimen preparations are suboptimal,e.g., in many clinical samples.

" Mark the area of interest on the backof the slide using a diamond stylus.

" Wash slide in 2 x SSC at 37°C for 5 min (in a Coplin jar).

" Apply 120 µl RN ase A solution(100 µg/ml RN ase A in 2 x SSC) toslide, cover with larger coverslip (e.g.22 x 50 mm), and incubate for 1 h at37°C in a moist chamber.

" Wash slide in 2 x SSC (or PBS) for 3 x5 min (Coplin jar).

" Wash slide in prewarmed PBS for 5 min at 37°C.

VI. Troubleshooting guide for in situ hybridization on chromosome spreadsfrom Stefan Joos and Peter Lichter, GermanCancer Research Center, H eidelberg,Germany.

Use the following tips to diagnose andcorrect commonly occurring problems inthe FISH protocols described above.

A. If no signal is observed, then:!1 Amplify signal.!2 Check probe labeling by performing the

following dot blot assay:" Spot serial dilutions of labeled

control." Spot serial dilutions of labeled sample." Compare intensities of the spots.

Note: For the detailed procedure onhow to estimate the labeling efficiencyplease refer to Chapter 4, IX , page 51.

!3 After labeling the probe according toSteps 1–4 of the nick translationprocedure (Procedure III) in Chapter 4of this manual, check the size of thelabeled probe molecules as follows:" To a 5–10 µl aliquot of probe, add gel

loading buffer and denature the probeby incubating it in a boiling waterbath for 3 min, then cool it on ice for3 min.Note:Keep the remainder of the probesample on ice while running the gel.

" Load the aliquot on a standard 1–2%agarose minigel along with a suitablesize marker.

" Q uickly run the gel (e.g. 15 volts percm for 30 min) to avoid renaturationof the probe in the gel.

" Visualize DN A in the gel, e.g. bystaining gel in 0.5 µg/ml ethidiumbromide, and take photograph duringUV illumination.Note: The probe molecules w ill be v isible as a smear. The smear shouldcontain only fragments smaller than500 nucleotides (nt) and larger than100 nt. A peak intensity at 250–300 nt seems optimal.

" Depending on the size of the probemolecules, do one of the following:I f the DN A is between 100–500 nt,proceed with the EDTA inactivationof the reaction mixture (as in Step 7 ofthe nick translation procedure,Chapter 4).

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D. If there is a starry backgroundfluorescence, then: %

" Incubate slide in pepsin solution (10 mg pepsin in 100 ml 10 mM H Cl)for 10 min at 37°C (in a water bath).

" Wash slide in PBS for 5 min at roomtemperature (RT).

" Incubate slide for 10 min at RT in asolution of 1% acid-free formalde-hyde, PBS, 50 mM MgCl2.

" Wash slide again in PBS for 5 min atRT.

" Dehydrate slide in a series of ethanolbaths (70% , then 90% , then 100% ; ≥ 5 min each) and air dry.

!6 Check quality of the formamide.Deionize formamide (several batchesfrom several sources should be tested)using ion exchange resin (e.g. DonexXG8).

!7 Check microscope.Be sure that mercury (H g2) lamp of themicroscope is in good condition andwell adjusted. O ld lamps don’t havegood excitation properties.

!8 Perform control experiments withalphoid DN A probes.

B. If the hybridization signal fades, then:!1 Change to a different batch of fluoro-

chrome-conjugated antibody.!2 Amplify with a secondary and/or

tertiary antibody to increase the signal.Note: Be aware of the background,which will also be amplified. Beforeamplification, remove embedding mediumby washing (2 x SSC, 37°–42°C, 4 x 10min).

!3 Try different antifading solutions [e.g.1,4-diazobicyclo-(2,2,2)-octane (DAPCO )or Vectastain (Vector)].

C. If there is a milky backgroundfluorescence, then: %

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Possible cause Remedy

Inadequate quality of Treat with pepsin to remove cell debrischromosome preparation (see protocol in Step A5 above).

Insufficient blocking Try to block with different reagents such as 3% BSA,human serum, or 3–5% dry milk.

Dirty glass slides Wash the slide with ethanol and rinse with water beforespreading chromosomes.

Bad quality of embedding Change embedding medium.medium

Possible cause Remedy

Agglutination of Perform a control experiment in the absence of fluorochromes probe to see if you get a non-specific signal from coupled to anti- the fluorochrome-conjugated antibody alone.bodies I f you do, spin the detection solution briefly and take

only the supernatant.Alternatively pass the detection solution through a G-50 Q uick Spin column to remove the agglutinates.

Labeled probe Check the size of the probe as described in Step A3 molecules are above and label again.too long

E. If there is a general strong staining ofchromosomes and nuclei, then:" Check the size of the labeled probe (as in

Step A3 above). It is most likely toosmall.

" I f the probe is too small, repurify (toremove any contaminating DN ase) andrelabel the probe.

ReferencesBentz, M.; Cabot, G.; Moos, M.; Speicher, M. R.,Ganser, A.; Lichter, P.; Döhner, H. (1994) Detectionof chimeric BCR-ABL genes on bone marrowsamples and blood smears in chronic myeloid andacute lymphoblastic leukemia by in situhybridization. Blood 83, 1922–1928.Dauwerse, J. G.; Wiegant, J.; Raap, A. K.; Breuning,M. H.; Van Ommen, G. J. B. (1992) Multiple colorsby fluorescence in situ hybridization using ratio-labeled DNA probes create a molecular karyotype.Hum. Mol. Genet. 1, 593–598.Wienberg, J.; Jauch, A.; Stanyon, R.; Cremer, T.(1990) Molecular cytotaxonomy of primates bychromosomal in situ suppression hybridization.Genomics 8, 347–350.

By comparing the hybridization patterns oftest and control DN A, the changes in signalintensities caused by imbalances within theanalyzed tissue can be identified.

Thus, CGH provides a comprehensivepicture of all chromosomal gains and losseswithin a single experiment. In contrast tobanding analysis, CGH requires no prepa-ration of metaphase chromosomes fromthe cells to be analyzed (which in certaintumor types may be difficult or evenimpossible).

In many cases, only small amounts oftumor tissue are available for cytogeneticanalysis. Therefore it is advantageous tocombine CGH with universal PCRprotocols (as shown in Figures 4 and 5under “Results”) (Speicher et al., 1993).Both sequence independent amplification(“SIA”) (Bohlander et al., 1992) and“DO P-PCR” (degenerate oligonucleotideprimer PCR) (Telenius et al., 1992) can becombined with CGH .

As is shown in Figure 2, the DO P primercontains a central cassette of 6 degeneratednucleotides and is flanked by specificsequences at the 3' end (6 nucleotides) andat the 5' end (10 nucleotides). The ampli-fication procedure is divided into two steps.During the first step, five PCR cycles areperformed at low stringency conditions(annealing temperature, Ta = 30°C). Theseconditions allow a frequent annealing ofthe DO P primer, which is primarilymediated by the short specific sequence atits 3' end and by the adjacent central cassetteof degenerated nucleotides. Eventually apool of many different DN A sequences isgenerated which should represent a statis-tically distributed subpopulation of thegenomic DN A. During the second step (35cycles) the annealing temperature is raisedto 62°C, allowing amplification only afterspecific base pairing of the full length prim-er sequence. Therefore, all the sequencesthat have been synthesized in the first stepare now exponentially amplified, resultingin a large amount of DN A that can be usedas probe for CGH .

Recently, a new approach of fluorescence insitu hybridization was introduced, called“comparative genomic hybridization”(CGH ) (Kallioniemi et al., 1992), whichallows the comprehensive analysis ofchromosomal imbalances in entire genomes(Du Manoir et al., 1993; Joos et al., 1993;Kallioniemi et al., 1992; Kallioniemi et al.,1993). The principle of CGH is shown inFigure 1. The genomic DN A from cellpopulations to be tested, such as fresh orparaffin-embedded tumor tissue, is labeledwith modified nucleotides (e.g., biotinylateddUTP) and used as a probe for in situhybridization to normal metaphase chromo-somes. This probe is called “test DN A”. As an

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Detection of chromosomal imbalances using DOP-PCR and comparative genomic hybridization (CGH) Stefan Joos, Barbara Schütz , Martin Bentz , and Peter L ichter, German Cancer Research Center, H eidelberg, Germany.

results in signals on normal chromosomes (partial karyotype)

total genomic DNA from cells to analyze (test DNA) labeled with biotin

total genomic DNA from normal cells(control DNA) labeled with digoxigenin

co-hybridization(normal chromosomes)

detection

signals from hybridized test DNA signals from co-hybridized control DNADNA from:

cells withamplified DNAor polysomies

cells withdeletions or monosomies

Figure 1: Schematic illustration of “comparative genomic hybridi-zation” (CGH) for chromosomeanalysis. For explanation, see text.

internal control, genomic DN A derived fromcells with a normal karyotype is differentiallylabeled and hybridized simultaneously(“control DN A”). For detection of thehybridized test and control DN A, differentfluorochromes are used, and each isvisualized by epifluorescence microscopywith selective filters. H ybridization with gen-omic DN A results in a general staining of allchromosomes. H owever, if the tissueanalyzed harbors additional chromosomalmaterial (e.g., a trisomy, amplifications etc.),hybridization reveals higher signal intensitiesat the corresponding target regions of thehybridized chromosomes (Figure 1). Corre-spondingly, deletions in the test sample arevisible as lower signal intensities (Figure 1).

!5 Transfer the reaction tubes to a thermo-cycler and start the PCR. Use the follow-ing thermal profile:" Initial denaturation: 10 min at 94°C." 5 Cycles, each consisting of:

1 min denaturation at 94°C1.5 min annealing (low stringency) at30°C3 min temperature ramping, from30°C to 72°C3 min elongation at 72°C.

" 35 Cycles, each consisting of:1 min denaturation at 94°C1 min annealing (high stringency) at62°C3 min elongation at 72°C, with theelongation time increased by 1 secondevery cycle [i.e., the 1st high stringencycycle has an elongation time of 3 min;the 2nd, 3 min and 1 s; the 3rd, 3 minand 2 s, etc.]

" Final elongation: 10 min at 72°C.

Protocols for DO P-PCR and CGH analysisare described below. Amplified sequencescan be detected directly by visual inspectionusing an epifluorescence microscope(“Results” Figures 3 and 4). H owever, thecomprehensive analysis of gains and lossesof single chromosomes (or parts thereof)requires digital image analysis with sophis-ticated software programs. For furtherdescription of the evaluation of CGHexperiments as well as the different steps ofthe CGH protocol, see the literature (DuManoir et al., 1995; Kallioniemi et al., 1994;Lichter et al., 1994; Lundsteen et al., 1995;Piper et al., 1995).

I. Preparation and labeling of DOP-PCR products !1 Prepare a DN A template by isolating

genomic DN A from small amounts oftissue (Maniatis, Fritsch, and Sambrook,1986). O ne mg of tissue results in about1 µg of genomic DN A. Note: See the literature (Kawasaki, 1990;Speicher et al., 1993) for protocols onDN A isolation from smaller amounts oftissue (down to only a few hundred cells)or from paraffin-embedded material.

!2 For each reaction, mix the followingcomponents in a PCR reaction tube onice:" 5.0 µl 10 x concentrated DO P-PCR

buffer (20 mM MgCl2; 500 mM KCl;100 mM Tris-H Cl, pH 8.4; 1 mg/mlgelatin).

" 5.0 µl 10 x concentrated nucleotidemix (dATP, dCTP, dGTP, dTTP, 2 mM each).

" 1–10 µl genomic DN A (0.1–1 ng/µl)." 5.0 µl 10 x concentrated DO P-PCR

primer [5' -CCGACTCGAGN N N NN N ATGTGG-3' (N =A,C,G, or T),20 µM].

" enough sterile water to make a finalreaction volume of 50 µl.

" 0.25 µlTaq DN A Polymerase (5 units/µl) (should be added last).

!3 Prepare a negative control by combin-ing the same solutions as in Step 2, butomitting the template DN A.

!4 O verlay reaction mixtures with 50 µlmineral oil.

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Figure 2: Schematic illustration of DOP-PCR.For explanation, see text.

DOP-PCRTelenius et al., 1992

CCGACTCGAGNNNNNN ATGTGG 3´5´

3´5´

5´ 3´

Higher stringency PCR (Ta = 62°C; 35 cycles)➝ specific priming of pre-amplified sequences2.

Low stringency PCR (Ta = 30°C; 5 cycles)➝ frequent priming at multiple sites1.

!4 Co-precipitate the probe DN As in eachtube by adding 1/20 volume of 3 Msodium acetate and 2.5 volumes of100% ethanol. Mix well and incubate at–70°C for 30 min.

!5 Spin the precipitated DN A in amicrocentrifuge at 12,000 rpm for 10min at 4°C. Discard the supernatant andwash the pellet with 500 µl of 70%ethanol.

!6 Spin the precipitated DN A again (12,000rpm, 10 min, 4°C). Discard thesupernatant and lyophilize the pellet.

!7 Add 6 µl deionized formamide to thelyophilizate and resuspend the probeDN A by vigorously shaking the tubefor > 30 min at room temperature.

!8 Add 6 µl of hybridization buffer to thetube containing probe DN A and againshake for > 30 min.

!9 Denature and dehydrate normal meta-phase chromosomal DN A on slides asdescribed in the literature (Lichter andCremer; Lichter et al., 1990).

!10 Prepare the probe (from Step 8) forhybridization by performing thefollowing steps:" Denature probe DN A at 75°C for

5 min. " Preanneal repetitive sequences by

incubating the probe at 37°C for20–30 min.

!11 Add prepared probe (from Step 10) todenatured chromosomal spreads (fromStep 9). Proceed with in situ hybridi-zation and probe detection as describedin the literature (Lichter and Cremer;Lichter et al., 1990).

!12 Evaluate the CGH experiment with anepifluorescence microscope.

ResultsThe CGH procedure was used to revealchromosomal imbalances (Figure 3) in a T-cell prolymphocytic leukemia (T-PLL).Chromosomal regions 6q, 8p, and 11qterare stained more weakly in the tumorDN A than in the control DN A, indicatingdeletions of all or part of the chromosomalarms in the tumor DN A. CGH alsoidentified overrepresented chromosomalregions (6p, 8q, and 14q) which stain moreheavily in the tumor than in the control.

!6 Analyze aliquots of the reaction mix-tures on an agarose gel by performingthe following steps:" Mix the aliquots with gel loading

buffer (0.25% bromophenol blue and30% glycerol).

" Load samples on a minigel. As a sizemarker use a 1 kb ladder or othersuitable size standard.

" Run the gel in 1x TBE buffer (89 mMTris base, 89 mM boric acid, 2 mMEDTA; pH 8.0) for 30 min at 100volts.

!7 Stain the gel with ethidium bromide andphotograph. In the reaction samples, youshould see a smear of DN A, ranging insize from about 200 bp to 2000 bp. N osmear should be visible in the negativecontrol.

!8 Collect the amplified DN A productsfrom the reaction mix by using the H ighPure PCR Product Purification Kit, seepage 50.

!9 Label the amplified DN A by standardnick translation procedures (Lichter andCremer; Lichter et al., 1994).

II. Comparative genomic hybridization (CGH) !1 Prepare slides of normal metaphase

chromosomes as described in theliterature (Lichter and Cremer; Lichteret al., 1990).

!2 Prepare the following solutions: " 3 M sodium acetate, pH 5.2. " Deionized formamide (molecular

biology grade): For deionization, useion exchange resin. The conductivityof the formamide should be below100 µSiemens.

" H ybridization buffer (4 x SSC, 20%dextran sulfate): Prepare 20 x SSC.Carefully dissolve dextran sulfate inwater to make a 50% dextran sulfatesolution. Autoclave the dextran sul-fate solution or filter it through anitrocellulose filter. Mix 200 µl of20 x SSC, 400 µl of 50% dextran sul-fate and 400 µl of double-distilledwater. Store the hybridization bufferat 4°C until you use it.

!3 To prepare probe DN A, combine 1 µgof each labeled test and control DN Awith 50–100 µg of human C ot-1 DN A.

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5

The DO P-PCR and CGH proceduresdescribed in this article were used to detectamplified sequences in genomic DN A fromspinal fluid of patients who have a tumor ofthe central nervous system (Figure 4).DO P-PCR and CGH also revealed anamplification of the c-myc oncogene(which maps to chromosomal band 8q24)in cell line H L60 (Figure 5).

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Figure 3: Comparative genomichybridization (CGH) experimentrevealing chromosomal imbal-ances in a T-cell prolymphocyticleukemia (T-PLL).DNA from tumor cells was labeledwith biotin and detected via a FITC-labeled antibody (green) whereasthe control DNA was labeledwithdigoxigeninanddetected via arhodamine-labeled antibody (red). For explanation, see the text. This photograph was taken by S. Joos and P. Lichter withpermission of Springer Verlag,Heidelberg and New York.

Figure 4: Detection of amplifiedsequences in the genomic DNA ofpatients with a central nervoussystem (CNS) tumor.Genomic DNA was isolated from asmall number of cells harvestedfrom the spinal fluid of the patients.The DNA was amplified by DOP-PCRand analyzed by CGH as describedin the text. CGH reveals a strongamplification signal on both homo-logues of chromosome 8q (arrows).The image was photographeddirectly using a conventional cameraon an epifluorescence microscope.

!!

Figure 5: Detection of amplified c-myc oncogene sequences incell line HL60.Genomic DNA (equivalent to the DNA from only 3–4 HL60 cells) wasdiluted in TE buffer, amplified byDOP-PCR, and analyzed byCGHasdescribed in the text. After CGH, theamplification signal is clearly visibleon both chromosome 8 homologues(arrows). The image was capturedas a gray scale image using a CCDcamera (Photometrix), pseudocol-ored, and photographed directlyfrom the computer screen.

!

!

!

!

!

Reagents available from Boehringer Mannheim for these procedures

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Product Description Cat. No.DOP-PCR Master*, ** for 25 amplification reactions and 5 control reactions 1644 963

Reagent Description Available asDOP-PCR mix, 25 units Taq DNA Polymerase/500 µl; 3 mM MgCl2; Three vials No. 1,2 x concentrated dATP, dGTP, dCTP, dTTP, 0.4 mM each; 100 mM KCl; 3 x 500 µl

20 mM Tris-HCl; 0.01% (v/v) Brij® 35; pH 8.3 (20°C)DOP-PCR primer (Sequence, 40 µM in double distilled H2O One vial No. 2, 5'-CCG ACT CGA GNN NNN 150 µlNAT GTG G-3' (N = A, G, C,or T, in approximatelyequal proportions) Double distilled H2O, Two vials No. 3, 2 x 1000 µlPCR gradeControl DNA Human genomic DNA from the lymphoblastoid One vial No. 4,

cell line BJA, 1 ng/µl, in double distilled H2O 50 µl

The DOP-PCR Master contains:

The High Pure PCR Product Purification Kit contains:

Reagent Description Cat. No. Pack size DIG-Nick Translation 5x conc. stabilized reaction buffer in 50% 1745 816 160 µl Mix* for in situ probes glycerol (v/v)andDNAPolymeraseI, DNaseI,

0.25 mMdATP, 0.25 mMdCTP, 0.25 mMdGTP,0.17 mM dTTP and 0.08 mM DIG-11-dUTP.

Biotin-Nick Translation 5x conc. stabilized reaction buffer in 50% 1745 824 160 µl Mix* for in situ probes glycerol (v/v)andDNAPolymeraseI, DNase I,

0.25 mM dATP, 0.25 mM dCTP, 0.25 mM dGTP0.17 mM dTTP and 0.08 mM biotin-16-dUTP.

Streptavidin-Fluorescein 1 428 578 1 mgAnti-Digoxigenin-Rhodamine 1 207 750 200 µg

Other reagents:

Reagent Description Available as• Binding buffer, • nucleic acids binding buffer; 3 M guanidine-thiocyanate, • Vial 1

green cap 10 mM Tris-HCl, 5% (v/v) ethanol, pH 6.6 (25°C)• Wash buffer, • wash buffer; add 4 volumes of absolute ethanol • Vial 2

blue cap before use! Final concentrations; 20 mM NaCl,2 mM Tris-HCl, pH 7.5 (25°C), 80% ethanol

• Elution buffer • elution buffer; 10 mM Tris-HCl, 1 mM EDTA, • Vial 3pH 8.5 (25°C)

• High Pure filter tubes • Polypropylene tubes, containing two layers of a specially pre-treated glass fibre fleece; maximumsample volume: 700 µl

• Collection tubes • 2 ml polypropylene tubes

Product Description Cat. No.High Pure PCR Product Kit for 50 purifications 1732668Purification Kit** Kit for 250 purifications 1732676**This product or the use of this

product may be covered by one or more patents of BoehringerMannheim GmbH, including thefollowing: EP patent 0 649 909(application pending).

**This product is sold under licensing arrangements withRoche Molecular Systems and The Perkin-Elmer Corporation.Purchaseof this product is accom-panied by a license to use it in thePolymerase Chain Reaction (PCR)process in conjunction with anAuthorized Thermal Cycler. Forcomplete license disclaimer, seeinside back cover page ! + ".

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typically result in a starry background outside of the chromosomes. In contrast,probes which are too small produce ahomogeneous background distributed overall chromosomes. Smaller probes may alsolead to a homogeneously strong stainingpattern in regions which are over- orunder-represented in the tumor genome.

Problems which may hinder evaluation of CGH

In contrast to conventional FISH experi-ments, strong fluorescence on the chromo-somes and little or no fluorescence in theareas where no chromosomes or nuclei arelocated, are no guarantee that a hybridi-zation experiment will be useful for a CGHanalysis. Even in cases where a bright andsmooth staining is observed, other factorscan severely impair evaluation of thehybridization experiment. Such impair-ment has two principal causes:" Insufficient suppression of repetitive

sequences may lead to incorrect measure-ments of ratios in chromosomal regionswhich contain many highly variablerepetitive sequences, e.g., sequences onchromosome 19. Strong staining of theheterochromatin blocks on chromo-somes 1, 9, 16, and 19 can serve as anindicator for poor suppression. Note: Good suppression will lead to verylow FITC and rhodamine fluorescenceintensities in the heterochromaticchromosome regions. Consequently, evenvery small variations of fluorescenceintensities may cause gross ratio varia-tions within these chromosomal regions.Thus, they are excluded from evaluation.Remedy: Either increase the amount ofthe C ot-1 DN A fraction or use longerpreannealing times (the latter being lesseffective) to achieve adequate suppres-sion of signals in such regions of thegenome.

" N on-homogeneous illumination of theoptical field leads to considerable re-gional variations within the ratio image.Such variations make a quantitative eval-uation of the CGH experiment com-pletely impossible. Remedy: Center the light source of themicroscope carefully.

Troubleshooting CGH experimentsControl to detect CGH of insufficient quality

H ybridization of a DO P-PCR probe tonormal human male metaphase chromo-somes should show a markedly weakerstaining of the X chromosome than ofother chromosomes. If there is not amarked difference between the X chromo-some and other chromosomes, the controlindicates a defective or poor quality CGHexperiment.

Usually these poor hybridization resultsare accompanied by other hallmarks of poorquality, like a speckled, non-homogeneousstaining pattern or high background fluo-rescence, both of which may considerablydisturb the quantitative image analysis bycausing high variability within the ratioprofiles.

Such poor hybridization results can be dueto many causes. Several are listed below.

Potential problems with chromosomalpreparations

In our experience, the most importantfactor for good hybridization experiments is the careful preparation of metaphasespreads. Therefore, every new batch ofchromosomes needs to be tested. If goodhybridization results are not obtained,discard the whole batch and start a newslide preparation procedure.

Note: Although impaired hybridization ismainly due to residual cell debris on theslides, proteolytic digestion of the chromo-somes will not lead to high quality CGHexperiments. A lthough proteolytic digestionwill improve the hybridization pattern onsuboptimal slides, the results are consider-ably worse than those obtained onoptimum slides which have not beenpretreated with protease.

Potential problems with probes

Two important causes for granular andnon-homogeneous staining patterns are:" Probe DN A preparations may be con-

taminated with high amounts of protein." The size of the labeled probe fragments

after nick translation may be inappro-priate. Fragments which are too large

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Lichter, P.; Bentz, M.; du Manoir, S.; Joos, S. (1994)Comparative genomic hybridization. In: Verma, R;Babu S. (eds) Human Chromosomes. New York: McGraw Hill, 191–210.Lichter, P.; Cremer, T. Chromosome analysis by non-isotopic in situ hybridization. In: HumanCytogenetics: Constitutional Analysis. IRL Press. Lichter, P.; Tang, C. C.; Call, K.; Hermanson, G.;Evans, G. A.; Housman, D.; Ward, D. C. (1990) Highresolution mapping of human chromosome 11 by insitu hybridization with cosmid clones. Science 247,64–69. Lundsteen, C.; Maahr, J.; Christensen, B.; Bryndorf,T.; Bentz, M.; Lichter, P.; Gerdes, T. (1995) Imageanalysis in comparative genomic hybridization.Cytometry 19, 42–50.Maniatis, T.; Fritsch, E. F.; Sambrook, J. (1986)Molecular Cloning: A Laboratory Manual. ColdSpring Harbor, New York: Cold Spring HarborLaboratory Press. Piper, J.; Rutovitz, D.; Sudar, D.; Kallioniemi, A.;Kallioniemi, O.-P.; Waldmann, F. M.; Gray, J. W.;Pinkel, D. (1995) Computer image analysis ofcomparative genomic hybridization. Cytometry 19,10–26.Speicher, M. R.; du Manoir, S.; Schröck, E.; Holtgreve-Grez, H.; Schoell, B.; Lengauer, C.; Cremer, T.; Ried,T. (1993) Molecular cytogenetic analysis offormalin-fixed, paraffin-embedded solid tumors bycomparative genomic hybridization after universalDNA amplification. Hum. Mol. Genet. 2, 1907–1914.Telenius, H.; Pelmear, A. H.; Tunnacliffe, A.; Carter,N. P.; Behmel, A.; Ferguson-Smith, M. A.; Nordenskjöld, M.; Pfragner, R. (1992) Ponder BAJP:Cytogenetic analysis by chromosome painting usingDOP-PCR amplified flow-sorted chromosomes.Genes, Chromosomes and Cancer 4, 257–263.

Problems inherent in sample composition

Apart from the experimental factors listedabove, the validity of a CGH experimentcritically depends on the percentage of cellscarrying chromosomal imbalances. If nogains or losses of chromosomes are detected, the sample may contain only alow proportion of tumor cells. Therefore,adequate information from the histologicallaboratory is crucial for any type of CGHanalysis.

References Bohlander, S. K.; Espinosa, R.; Le Beau, M. M.;Rowley, J. D.; Diaz, M. O. (1992) A method for therapid sequence-independent amplification ofmicrodissected chromosomal material. Genomics13, 1322–1324. Du Manoir, S.; Schröck, E.; Bentz, M.; Speicher, M. R.; Joos, S.; Ried, T.; Lichter, P.; Cremer, T. (1995)Quantitative analysis of comparative genomichybridization. Cytometry 19, 27–41.Du Manoir, S.; Speicher, M. R.; Joos, S.; Schröck,E.; Popp, S.; Döhner, H.; Kovacs, G.; Robert-Nicoud,M.; Lichter, P.; Cremer, T. (1993) Detection ofcomplete and partial chromosome gains and losses by comparative genomic in situ hybridization. Hum. Genet. 90, 590–610. Joos, S.; Scherthan, H.; Speicher, M. R.; Schlegel, J.;Cremer, T.; Lichter, P. (1993) Detection of amplifiedgenomic sequences by reverse chromosomepainting using genomic tumor DNA as probe. Hum. Genet. 90, 584–589. Kallioniemi, A.; Kallioniemi, O.-P.; Sudar, D.;Rutovitz, D.; Gray, J. W.; Waldman, F.; Pinkel, D.(1992) Comparative genomic hybridization formolecular cytogenetic analysis of solid tumors.Science 258, 818–821. Kallioniemi, O-P.; Kallioniemi, A; Piper, J.; Isola, J.;Waldman, F.; Gray, J. W.; Pinkel, D. (1994)Optimizing comparative genomic hybridization foranalysis of DNA sequence copy number changes insolid tumors. Genes, Chromosomes and Cancer 10,231–243.Kallioniemi, O.-P.; Kallioniemi, A.; Sudar, D.;Rutovitz, D.; Gray, J. W.; Waldman, F.; Pinkel, D.(1993) Comparative genomic hybridization: a rapidnew method for detectingandmappingDNAamplification in tumors. Seminars in Cancer Biology4, 41–46.Kawasaki, E. S. (1990) Sample preparation fromblood, cells, and other fluids. In: Innis, M. A.;Gelfand, D. H.; Sninsky, J. J.; White, T. J. (eds) PCR Protocols: AGuidetoMethodsand Applications.San Diego, CA: Academic Press, 146–152.

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optimal fluorochrome for the PRIN Sreaction with the convenience of thePRIN S reaction. This combination pro-duces extremely rapid results as there is noconjugate incubation step required and thebest sensitivity in direct detection compar-able to indirect detection.

In the Rhodamine PRIN S Reaction Set,dTTP is provided at an optimized concen-tration in the Rhodamine Labeling Mixbecause dTTP variation has only minorinfluence on rhodamine labeling intensity.

PRimed IN Situ Labeling (PRIN S) is a fastalternative to traditional in situ hybridi-zation (Koch et al., 1989). PRIN S startswith in situ hybridization (annealing) of anunlabeled synthetic oligonucleotide (e.g.,one of the PRIN S oligonucleotide primersin Table 1) to denatured chromosomes inmetaphase spreads or interphase nuclei onmicroscope slides. Then, the hybridizedoligonucleotide serves as primer which athermostable DN A polymerase (Gosdenet al., 1991) elongates in situ. During theprimer elongation reaction, the polymeraseincorporates nonradioactively labeled nucleotides into the newly synthesizedDN A (Gosden and H anratty, 1993).

Depending on the label, the newly synthe-sized DN A may be detected by one of twomethods:Indirect detection using the DIG PRIN SReaction Set or direct detection using theRhodamine PRIN S Reaction Set.

! Indirect detection uses a fluorochrome-conjugated anti-DIG antibody (for example, Anti-Digoxigenin-Fluoresceinfrom the DIG PRIN S Reaction Set) tolocate digoxigenin-(DIG-)labeled DN A.

For indirect detection, we offer the DIGPRIN S Reaction Set, which combinesthe high sensitivity of digoxigenin label-ing with the power of the PRIN Sreaction. In the Digoxigenin PRIN SReaction Set the sensitivity of thePRIN S reaction can be optimized byadjusting the dTTP-concentration indi-vidually according to experimentalrequirements.

! Direct detection uses fluorochrome-labeled nucleotides (e.g. Rhodamine-dUTP from the Rhodamine-PRIN S Reaction Set) that are directly visibleunder a fluorescence microscope.

For direct detection, we offer theRhodamine PRIN S Reaction Set, whichcombines the labeling intensity of ourimproved rhodamine which is the

Identificationof chromosomesinmetaphasespreadsand interphase nuclei with PRINS oligonucleotideprimersandDIG- or rhodamine-labeled nucleotidesDr. R . Seibl, Research Laboratories, Boehringer Mannheim GmbH , Penzberg, Germany.

Table1:Specificity of PRINS oligo-nucleotide primers.

PRIN S oligo- Specific fornucleotide primer

1 Satellite II DN A in the pericentromeric hetero-chromatin on the long arm of human chromosome 1

3 Centromere of human chromosome 3, Alpha satellite



9 (Sat) Satellite III DN A in the pericentric heterochromatinon the proximal long arm of human chromosome 9




14 + 22 Centromeres of human chromosomes 14 and 22,Alpha satellite

1 + 16 Satellite II DN A of human chromosomes 1 and 16 inboth cases located to the proximal long arm of thatchromosome.



X Centromere of human chromosome X, Alpha satellite

Y Long arm of human chromosome Y. Stains most of the long arm of the Y-chromosome.

T Telomeres of all human chromosomes1

Control primer All human chromosomes

1 Please note the Telomere Primer is only for use with the Digoxigenin PRINS Reaction Set,because the Rhodamine PRINS Reaction Set contains dTTP in the Rhodamine Labeling Mix.

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I. Preparation of slides"1 Using standard methods (e.g. Gosden,

1994; Roonea and Czepulkowski, 1992),prepare fresh microscope slides contain-ing fixed metaphase chromosomes and/or interphase nuclei.Caution: For best results, prepare theslides containing the fixed metaphasechromosomes either immediately beforeuse or on the day before the PRIN Sreaction. I f slides have been prepared aday early, store them at 4°C until use.Such slides may be used in the PRIN Sreaction w ithout formamide pretreat-ment (Step 2 below).

"2 (O ptional) If slides have been stored for3 or more days at room temperature in70% ethanol at 4°C, denature the prep-arations with formamide directly beforethe PRIN S reaction. For the formamidetreatment, perform the following steps:! Prepare a solution containing:

– 70 ml deionized formamide– 10 ml 20 x SSC– 10 ml 500 mM sodium phosphate

buffer, pH 7.0– 10 ml H 2O

! Adjust the pH of the formamide solu-tion to 7.0 with H Cl.

! Incubate the slides for 45 s at 68°C inthe formamide solution. Check thetemperature of the formamide solu-tion, as it is important that it be68°C.

! Wash the slides in a series of ice-coldethanol solutions (70% , then 90% ,then 100% ethanol).Caution: The ethanol solutions mustbe ice-cold during this step.

Compared to traditional ISH methods(where probe labeling is performed beforethe probe is added to the slide), PRIN S hastwo advantages:

! Convenience. PRIN S circumvents thelaborious probe isolation and labelingneeded in other approaches.

! Speed. Since high probe concentrationsare possible, hybridization and chainelongation (in one reaction) usually takes30 min. For many PRIN S primer thereaction time can even be reduced. Thecomplete procedure, including fluores-cent antibody detection, is complete inapproximately 2 h. When using theRhodamine PRIN S Reaction Set fordirect detection results are already avail-able after 60 min.

This article describes the use of the PRIN SReaction Set, the Rhodamine-PRIN SReaction Set, and PRIN S oligonucleotideprimers to rapidly identify human chromo-somes. It also details how to combinePRIN S and FISH (fluorescence in situhybridization) for a multiple labeling experiment.

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! The dTTP concentration can beadjusted indiv idually to increase thesignal strength by reducing the dTTPconcentration or to decrease thesignal strength by increasing thedTTP concentration.

! 2.5 µl Taq DN A Polymerase (1 unit/µl).

B. I f you want to use rhodamine-labeledDN A and detect the PRIN S target bydirect fluorescence methods, prepare 30 µl of Rhodamine-PRIN S reactionmix (for each reaction) by adding thefollowing components to a sterilemicrocentrifuge tube (on ice):Note: Most of the components (num -bered v ials) used in the Rhodamine-PRIN S reaction mix are from theRhodamine-PRIN S Reaction Set.

! 16.5 µl sterile redistilled H 2O .! 3.0 µl 10 x concentrated PRIN S

reaction buffer (Rh-PRIN S vial 2).! 3.0 µl 10 x concentrated Rhodamine-

PRIN S labeling mix (500 µM each ofdATP, dCTP, and dGTP; 50 µMdTTP; 10 µM rhodamine-dUTP; 50%glycerol) (Rh-PRIN S vial 1).Note: The correct dTTP concentration(5 µM) for most primers is provided as part of the Rhodamine-PRIN Slabeling mix (in the Rhodamine-PRIN S Reaction Set). Variations indTTP do not significantly affect theintensity of rhodamine labeling.

! Since dTTP is included in theRhodamine-PRIN S labeling mix andbecause for the PRIN S O ligonucleo-tide Primer T, no dTTP has to be addedto the reaction mix, the T primer isincompatible with the components ofthe Rhodamine-PRIN S Reaction Set.

! 5.0 µl PRIN S oligonucleotide primer[either control primer (Rh-PRIN Svial 3) or a human chromosome-specific PRIN S oligonucleotideprimer (Table 1).

! 2.5 µl Taq DN A Polymerase (1 unit/µl)."2 Gently vortex the reaction solution to

mix the components."3 Centrifuge the reaction mixture briefly

to collect the liquid on the bottom ofthe tube.

II. Preparation of PRINS reaction mixesNote: For a single reaction under a 22 x22 mm coverslip, prepare 30 µl of DIG-PRIN S reaction mix or Rhodamine-PRIN Sreaction mix. Adjust the volume of thereaction mix (but not the concentration ofthe components) if you are preparing morethan one reaction, or if you use coverslipsof a different siz e. For 18 x 18 mm cover-slips, prepare 15 ml reaction mix perreaction; for 24 x 60 mm or 22 x 40 mmcoverslips, prepare 60 ml per reaction."1 Depending on the type of PRIN S

reaction desired, do one of the following:

A. I f you want to use Digoxigenin for insitu labeling and detect the PRIN S targetby indirect fluorescence (immunological)methods, prepare 30 µl of DIG-PRIN Sreaction mix (for each reaction) byadding the following components to asterile microcentrifuge tube (on ice):Note: Most of the components (num-bered v ials) used in the DIG-PRIN Sreaction mix are from the PRIN SReaction Set.

! 13.5 µl sterile redistilled H 2O .! 3.0 µl 10 x concentrated PRIN S

reaction buffer (PRIN S vial 3).! 3.0 µl 10 x concentrated DIG-PRIN S

labeling mix (500 µM each of dATP,dCTP, and dGTP; 50 µM DIG-dUTP; 50% glycerol) (PRIN S vial 1).

! 5.0 µl PRIN S oligonucleotide primer[either control primer (PRIN S vial 4) ora human chromosome-specific PRIN Soligonucleotide primer (Table 1).

! 3.0 µl 450 mM dTTP (PRIN S vial 2).Caution: The concentration of dTTPused in the DIG-PRIN S reactionmix depends upon the PRIN S oligo-nucleotide primer used. Using the PRIN S O ligonucleotidePrimer T, no -dTTP has to be addedto the reaction mix. For the PRIN S O ligonucleotide Primer9, the final concentration of dTTP inthe DIG-PRIN S reaction mix must be90 µM. For all other PRIN S primers listed inTable 1 the final concentration ofdTTP in the DIG-PRIN S reactionmix must be 45 µM. The correct dTTP concentration forother primers must be determinedempirically.

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! I f slides are 3 or more days old andhave been treated with formamide,skip this step, leave the heating blockset at 60°C and go to Step 5.

"5 Reduce the temperature of the heatingblock to 60°C and incubate the slidesfor 30 min at 60°C while the primeranneals and the polymerase elongates it.Caution: Be sure the temperature of theslides is exactly as indicated for theindiv idual primers. Even a few secondsof exposure to a temperature below thestringent annealing temperature canlead to an unwanted background signal.The slides are usually at the correcttemperature when the surface of theheating block reaches 60°C.

Note: The stringent annealing/ elongationtemperature for the PRIN S reactiondepends on the sequence of the primerused.The annealing temperature in the PRIN Sreaction using the O ligonucleotidePrimer T is 60°C.The annealing temperature for all otherPRIN S O ligonucleotide Primer in thePRIN S reaction is 60°–65°C.

"6 Remove the slides from the heatingblock and place them immediately in aCoplin jar containing stop buffer whichhas been prewarmed to the temperatureof the hybridization/elongation reaction(60°C).

"7 Stop the reaction by washing the slidesfor 3–5 min at the temperature of the hy-bridization/elongation reaction (60°C)with prewarmed stop buffer.Note: Coverslips should drop off theslides during this step.

Caution:Be sure the temperature remainsthe temperature of the hybridization/elongation reaction (60°C) throughoutthe wash.

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III. Primer annealing and elongation"1 Place the dry slides containing fixed

chromosomes and/or interphase nucleion either a heating block or a specialthermal cycler for slides.Note: To guarantee specificity andsensitiv ity of the reaction, the denatura-tion step and the annealing/elongationstep need precisely controlled elevatedtemperatures. For reproducible results,we recommend that, instead of a heatingblock , you use a temperature cycler thatis specially designed for slides, such as theO mniSlide or O mniGene TemperatureCycler (H ybaid L td., Teddington,Middlesex, England) or an equivalentinstrument from another manufacturer.

"2 Depending on the age of the slides, doone of the following:! I f slides are freshly prepared (one day

old or less), incubate them at 94°Cfor 1 min.

! I f slides are 3 or more days old andhave been treated with formamide(Procedure I, Step 2 above), incubatethem at 60°C for 1 min.

"3 Without removing the slides from theheating block, do the following:! O nto each sample, immediately

pipette:– 25 µl of DIG-PRIN S reaction mixor– 27 µl of Rhodamine-PRIN S

reaction mix! Cover sample with a 22 x 22 mm

coverslip.Caution: I f you use coverslips of adifferent siz e than 22 x 22 mm, adjustthe volume of the DIG-PRIN S orRhodamine-PRIN S reaction mix inthis step so it covers the sample welland completely wets the coverslip.

"4 Depending on the age of the slides, doone of the following:! I f slides are freshly prepared (1 day old

or less), incubate them at 91°–94°C for3 min to denature the chromosomalDN A, then go to Step 5.Caution: The slides generally reach91°–94°C when the surface of a heat-ing block reaches 94°–97°C . H ow-ever, if you are using a temperaturecycler designed for slides, the cyclermay automatically take into accountthis temperature differential.

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IV. Detection of hybridized sequences"1 In a Coplin jar, wash the slides as follows:

! 3 x 5 min at 37°C with wash buffer[0.2% Tween® 20 in phosphate bufferedsaline (PBS)].

! About 1 min with PBS at roomtemperature.

"2 Depending on the label used in thePRIN Sreaction, do oneof the following:! I f you produced DIG-labeled DN A

in Procedure III, go to Step 3.! I f you produced rhodamine-labeled

DN A in Procedure III, go to Step 6."3 Prepare the antibody working solution

by diluting the Anti-Digoxigenin-Fluorescein conjugate (PRIN S vial 5)1:1000 with PBS containing 1% block-ing solution.Note: Anti-DIG-Fluorescein conjugate isa included in the PRIN S Reaction Set.10 x blocking solution is included in theDIG Wash and Block Set*; it contains100 mM maleic acid (pH 7.5), 150 mMN aCl, and 10% blocking reagent. PBScontaining 1% blocking solution can beprepared by diluting the 10 x blockingsolution 1:10 with PBS.

"4 In a Coplin jar, incubate the slides withantibody working solution for 30 minin the dark at 37°C.

"5 Repeat Step 1 washes."6 For counterstaining, add the counter-

stain working solution (e.g., PBScontain-ing either 20 ng/ml propidium iodide or20 ng/ml DAPI) to the Coplin jar andincubate the slides with the counterstainfor 5 min in the dark at room temperature.Caution: I f the target DN A is labeledwith rhodamine-dUTP, do not usepropidium iodide counterstain, since theemission of propidium iodide and rhoda-mine overlap. Use DAPI as a counter-stain for rhodamine-labeled DN A.! Wash the slides for 2–3 min under

running water. "7 Air-dry the slides in the dark."8 Prepare the slides for viewing by doing

the following:! Apply 20 µl antifade solution [e.g.,

Vectashield (Vector) or Slow Fade-Light (Molecular Probes)] to eachslide.

! Add a coverslip.Note: For long term storage of the slidesseal the edges of the coverslip with nailpolish and store in the dark at –20°C.

"9 Analyze the slides under a fluorescencemicroscope with the appropriate filters.

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V. Combining PRINS and FISH"1 Label a DN A probe with digoxigenin,

biotin, or any fluorochrome (except theone used in the PRIN S procedure),according to procedures described inChapter 4 of this manual.

"2 Perform the PRIN S reaction as describedin Procedure I–III of this article.

"3 After washing the PRIN S slides in stopbuffer (Procedure III, Step 6), dehydratethe slides in an ethanol series (increasingconcentrations of ice-cold ethanol).Caution: The ethanol solutions must beice-cold during this step.

"4 H ybridize the labeled DN A probe(from Step 1) to the PRIN S slides,according to FISH procedures describedelsewhere (Procedure IIA, IIB, or IICfrom the article “In situ hybridization tohuman metaphase chromosomes usingDIG-, biotin-, or fluorochrome-labeledDN A probes and detection with fluoro-chrome conjugates”, in Chapter 5 of thismanual, pages 62, 63). Modify thehybridization procedure as follows:! Denature the labeled probe DN A

before adding it to the slides.! Do not heat the slides to 80°C after

adding the probe to the slides."5 After the incubation step, wash the slides

with the formamide/SSC washes describedin the FISH procedure, then perform afinal wash in PBS.

"6 Depending on the label used in thePRIN Sreaction, do one of the following:! For PRIN S targets labeled with DIG-

dUTP, perform the PRIN S antibodydetection and wash steps as above(this article, Procedure IV, Steps 3–5).

! For PRIN S targets labeled withrhodamine-dUTP, skip this step andgo to Step 7.

"7 Depending on the label used in the FISHprocedure, do one of the following:! For biotin- or digoxigenin-labeled

FISH probes, perform the immuno-logical detection step according toprocedures described elsewhere (Pro-cedure IIIA1/ IIIA2 or IIIB1/ IIIB2from the article “In situ hybridizationto human metaphase chromosomesusing DIG-, biotin-, or fluorochrome-labeled DN A probes and detectionwith fluorochrome conjugates”, inChapter 5 of this manual, page 64).

! For fluorochrome-labeled FISHprobes, skip this step and go to Step 8.

*Sold under the tradename of Geniusin the US.

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"8 Counterstain and prepare the slides asabove (this article, Procedure IV, steps6–7).

"9 Analyze the slides under a fluorescencemicroscope with the appropriate filters.

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Possible cause Possible solution

Denaturation temperature does not reach Increase the temperature of the block 91°–94°C at the surface of the slide. slightly during denaturation.

Annealing/elongation temperature Lower the annealing/elongation temperatureis too high. by 3°C and repeat experiment. If signal is

inadequate, reduce the temperature further(in 3°C steps).

(For DIG-labeling only) N ot enough 1. Reduce the dTTP concentration in the DIG-dUTP was incorporated into reaction solution to 30 µM, 15 µM or zero sample. by adding 2 µl, 1 µl, or no dTTP solution

(DIG PRIN S Reaction Set vial 2) to each 30 µl reaction

Primer concentration too low 1. Increase the amount of primer in thereaction solution.Use the control primer (DIG PRIN SReaction Set vial 4) to check the sensitivityof the reaction.

B. If signal is strong, then:


(For DIG-labeling only) Too much 1. Increase the dTTP concentration in the DIG-dUTP was incorporated into reaction solution to 60 µM, 90 µM or sample. 135 µM by adding 4 µl, 6 µl, or 9 µl dTTP

solution (DIG PRIN S Reaction Set vial 2)to each 30 µl reaction

Primer concentration too high 1. Decrease the amount of primer in thereaction solution.Use the control primer (DIG PRIN SReaction Set vial 4) to check the sensitivityof the reaction.

VI. Troubleshooting guide for PRINSUse the following tips to diagnose andcorrect commonly occurring problems inthe PRIN S procedure described above.

A. If signal is weak or missing, then:

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5

C. If chromosomes or nuclei containunwanted background signals, then:

85



The slide was exposed to temperatures Do not allow the temperature on the slide below the stringent annealing/ to ever fall below the stringent annealing/ elongation temperature for a few elongation temperature.seconds after the denaturation step.

Annealing/elongation temperature is Raise the annealing/elongation temperaturetoo low. by 3°C and repeat experiment. If background

is still high, increase the temperature further(in 3°C steps) until the reaction is specific.

The slide was exposed to temperatures Prewarm the stop buffer to the stringent below the stringent annealing/elonga- annealing/elongation temperature andtion temperature for a few seconds transfer the slides directly from the heatingbetween the elongation step and the block to the warm stop solution.addition of stop buffer.

Primer-independent elongation of nicks Perform a control PRIN S reaction withoutand chromosomal breaks occurred. added primer. If you obtain a signal, eitherSuch nicks and breaks can occur during block the 3' ends of the chromosomal breakslong term storage of the chromosomal by incubating the chromosomal preparationspreparation. with DN A polymerase and a mixture of

ddN TPS or (preferably) discard thechromosomal preparations and prepare freshchromosomes or fresh chromosomal spreads.

(For DIG-labeling only) When the Increase the dTTP concentration in the primary signal is strong, minor primer- reaction solution to 60 µM, 90 µM ordependent signals may be seen at 135 µM by adding 4 µl, 6 µl, or 9 µl secondary binding sites for the primer. dTTP solution (DIG PRIN S Reaction Set

vial 2) to each 30 µl reactionorDecrease the amount of primer in the reactionsolution.

D. If the entire slide (not just the chromosomes and nuclei) contain unwanted background signals, then:


(For DIG-labeling only) The antibody Perform a control detection reaction by conjugate (anti-digoxigenin-fluores- incubating an unhybridized sample slide cein) bound nonspecifically to the slide directly with the antibody conjugate, then or to cellular proteins and matrix in washing and analyzing the slide as in the chromosomal preparation. Procedure IV. I f background is observed,

either increase the number and duration of the washing steps or (preferably) repeat thechromosomal preparation to remove excesscellular proteins and matrix responsible fornonspecific binding.

DIG-dUTP or rhodamine-dUTP Perform a control PRIN S reaction in the bound nonspecifically during the absence of Taq polymerase and primer. PRIN S reaction. I f background is observed, either increase

the number and duration of the washes or(preferably) repeat the chromosomalpreparation to remove excess cellular proteinsand matrix responsible for nonspecific binding.

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5

ReferencesGosden, J. R., ed. (1994) Chromosome AnalysisProtocols (Methods Mol. Biol., Vol. 29). Totowa, NJ:Humana Press.Gosden, J.; Hanratty, D.; Starling, J.; Fantes, J.;Mitchell, A.; Porteus, D. (1991) Oligonucleotideprimed in situ DNA synthesis (PRINS): A method forchromosome mapping, banding and investigation ofsequence organization. Cytogenet. Cell Genet. 57,100–104.

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Results

a b

Figure 1: PRINS labeling and indirect fluorescent detection of human chromosomes.The PRINS primers specific for (Panel a) human chromosome11 and(Panel b)human chromosome 7 werehybridized to human chromosomal preparations. The hybridized primers were elongated in situ and thenewly synthesized DNA labeled with DIG-dUTP according to the procedure described in the text. LabeledDNA was visualized with anti-DIG-fluorescein. Propidium iodide was used as a counterstain.

Figure 2: Rhodamine PRINS labeling and directfluorescent detection of human chromosomes.The PRINS primers specific for (Panel a) humanchromosome 1 and (Panel b1, b2) humanchromosome 9 were hybridized to humanchromosomal preparations. The hybridized primerswere used to label target DNA in situ withrhodamine-dUTP according to the proceduredescribed in the text. Labeled DNA was vieweddirectly under a fluorescence microscope. DAPI wasused as a counterstain.

a

b1

b2

Gosden, J.; Hanratty, D. (1993) PCR in situ: A rapid alternative to in situ hybridization formapping short, low-copy number sequenceswithout isotopes. BioTechniques 15, 78–80.Koch, J. E.; Kølvraa, S.; Petersen, K. B.; Gregersen,N.; Bolund, L. (1989) Oligonucleotide primingmethods for the chromosome-specific labeling ofalpha satellite DNA in situ. Chromosoma (Berl.) 98,259–265.Roonea, D. E.; Czepulkowski, B. H., eds. (1992)Human Cytogenetics, A Practical Approach, Vol. 1.Oxford, England: IRL Press.

#

$#

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* Please note the Telomer Primer isonly for use with the DigoxigeninPRINS Reaction Set because theRhodamine PRINS Reaction Setcontains dTTP in the Labeling Mix.

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Primer Cat. No. PRINS oligonucleotide primer 1, specific for human chromosome 1 1695 959PRINS oligonucleotide primer 1+16, specific for human chromosome 1+16 1768 549PRINS oligonucleotide primer 3, specific for chromosome 3 1695 967PRINS oligonucleotide primer 7, specific for human chromosome 7 1695 975PRINS oligonucleotide primer 8, specific for human chromosome 8 1695 983PRINS oligonucleotide primer 9 (Sat), specific for human chromosome 9 1768 565PRINS oligonucleotide primer 10, specific for human chromosome 10 1768 522PRINS oligonucleotide primer 11, specific for human chromosome 11 1695 991PRINS oligonucleotide primer 12, specific for human chromosome 12 1696 009PRINS oligonucleotide primer 14+22, specific for human chromosome 14+22 1768 557PRINS oligonucleotide primer 17, specific for human chromosome 17 1696 017 PRINS oligonucleotide primer 18, specific for human chromosome 18 1696 025PRINS oligonucleotide primer X, specific for the human X chromosome 1696 033 PRINS oligonucleotide primer Y, specific for the human Y chromosome 1696 041 PRINS oligonucleotide primer T, specific for human telomeres 1699 199 PRINS control oligonucleotide primer, specific for all human chromosomes 1699 172

Reagent Description Kit component 10 x Concentrated dNTPs including digoxigenin-11-dUTP One vial No. 1, 135 µlPRINS labeling mix dTTP solution 450 µM dTTP One vial No. 2, 200 µl10 x Concentrated Buffer for 45 reactions One vial No. 3, 150 µlPRINS reaction bufferPRINS Control Primer for 5 control reactions One vial No. 4Oligonucleotide Primer Anti-Digoxigenin-Fluorescein Antibody (200 µg/ml) for 22 detection Two vials No. 5,Conjugate, Fab fragments reactions in a Coplin jar 2 x 550 µl

PRIN S oligonucleotide primers The following PRIN S oligonucleotide primers are available* (Pack size 100 µl each):

Product Description Cat. No. DIGPRINS Reaction Set For 40 PRINS labeling reactions and 5 control reactions 1695 932

The DIG PRIN S Reaction Set includes a detailed working procedure and contains:

Reagent Description Kit component 10 x Concentrated Rhodamine dNTPs including Rhodamine-dUTP One vial No. 1, 135 µlPRINS labeling mix 10 x Concentrated Buffer for 50 reactions One vial No. 2, 150 µlPRINS reaction bufferPRINS Control Primer for 5 control reactions One vial No. 3, 25 µlOligonucleotide Primer

Product Description Cat. No. Rhodamine PRINS Reaction Set For 40 PRINS labeling reactions and 5 control reactions 1768 514

The Rhodamine PRIN S Reaction Set includes a detailed working procedure and contains:

Reagents available from Boehringer Mannheim for PRINS

I. Preparation of metaphase chromosomes!1 Prepare chromosomes from either whole

blood or cell culture as follows:" For whole blood, do these steps:

" Mix 5 ml whole blood with 5 ml PBS." Pipette 7.5 ml of lymphocyte separa-

tion medium into a 50 ml centrifugetube.

" Carefully layer blood-PBS mixtureatop separation medium.

" Centrifuge for 30 min at 800 x g." Remove the interphase (middle part

of the centrifuged mixture) that con-tains the lymphocytes.

" Place the lymphocytes in a freshcentrifuge tube.

" Mix lymphocytes with 50 ml PBS." Centrifuge for 10 min at 420 x g." Discard the supernatant and resuspend

cells at approximately 106 cells per mlin chromosome medium (Gibco).Note: This step should require about5 ml chromosome medium.

" Go to Step 2." For cells in culture, do these steps:

" H arvest cells by centrifugation." Resuspend cells at approximately 106

cells per ml in chromosome medium." Go to Step 2.

!2 Incubate cells in chromosome mediumfor 71 h in a CO 2 incubator at 37°C and7% CO 2.

!3 Add 12 µl Colcemid® per ml medium.!4 Incubate cells for 1 h at 37°C and 7%

CO 2.!5 Centrifuge cells (210 x g) for 10 min at

4°C.!6 With a pipette, remove supernatant until

only 1 ml remains atop the cells.

Fluorescence in situ hybridization (FISH )with human chromosome-specific satelliteDN A probes is a sensitive technique foridentifying individual human chromo-somes. Applications include:" Analysis of aneuploidies in normal or

tumor cells" Identification of chromosomes in

hybrid cells" Production of genetic linkage maps

Especially useful for such applications arefluorescein- or digoxigenin-labeled probeswhich recognize alphoid sequence variantsthat occur at the centromere of specificchromosomes (Willard and Waye, 1987;Choo, K. H . et al., 1991). Fluorescein-labeled probes allow rapid, direct detectionof a single chromosome. DIG-labeledprobes allow sensitive, indirect detectionof a chromosome with a variety offluorochrome-labeled antibodies. Bothfluorescein- and DIG-labeled probes canbe combined in a multicolor labeling ofdifferent chromosomes on a singlepreparation.

This article describes the use of such pre-labeled probes to identify human chromo-somes in chromosomal spreads from wholeblood or from cell culture.

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Identification of chromosomes in metaphasespreads with DIG- or fluorescein-labeled humanchromosome-specific satellite DNA probesDr. G . Sagner, Research Laboratories, Boehringer Mannheim GmbH , Penzberg, Germany.

III. In situ hybridization with chromo-some-specific DNA probesNote: Perform the following procedure ina 50 ml Coplin jar which can hold amaximum of 8 slides.

Caution: Prewarm all solutions in thefollowing procedure to the appropriatetemperature before using them.!1 Remove slides from the 70% ethanol

storage solution (from Procedure II,Step 7).

!2 Air dry slides completely (approxi-mately 30 min).

!3 For each slide, prepare 20 ml hybridi-zation solution containing:" 10–13 µl deionized formamide (final

concentration: 50–65% formamide)Note:Different probes require differentconcentrations of deionized formamide.See Table 1 for the correct formamideconcentration to use with each probe.

" 5 µl 4 x concentrated hybridizationbuffer [8 x SSC; 40% (v/v) dextransulfate; 4 mg/ml MB-grade DN A(from fish sperm); pH 7.0].

" 1–2 µl (20–40 ng) human chromo-some-specific satellite DN A probe,either fluorescein- or DIG-labeled.

" Enough redistilled H 2O to make atotal volume of 20 µl.Caution:Twenty µl is enough hybrid-ization solution if you are using a 24x 24 mm coverslip in Step 7 below. I fyou use a different siz e coverslip,adjust the volume of the hybridiza-tion solution (but not the concen-tration of the components) accordingly.

!4 Preheat a glass tray big enough to holdall slides in a 72°C oven.

!5 Prewarm a moist chamber to 37°C.!6 Pipette 20 µl hybridization solution

onto each slide, add a 24 x 24 mm cover-slip, and seal the coverslip to the slidewith rubber cement.

!7 Place the prepared slides onto the pre-warmed glass tray in the oven and incu-bate 5–10 min at 72°C to denatureDN A.

!8 Transfer slides to the prewarmed moistchamber and incubate overnight at37°C.

!7 Resuspend the pellet in the remainingsupernatant.

!8 Slowly add to the resuspended cells10mlof prewarmed (37°C) 75 mM KCl.

!9 Gently mix and incubate for 12–20 minat 37°C.

!10 To the mixture, add 7 drops of chilledmethanol-acetic acid fixative (3 partsmethanol to 1 part acetic acid, precooledto –20°C). Gently mix.

!11 Centrifuge mixture (210 x g) for 10 minat 4°C.

!12 With a pipette, remove supernatant untilonly 1 ml remains atop the pellet.

!13 Carefully resuspend the pellet in theremaining supernatant and add 1 mlprecooled (–20°C) methanol-acetic acidfixative. Gently mix.

!14 Add an additional 5–10 ml precooled(–20°C) methanol-acetic acid fixative.Mix thoroughly.

!15 Fix chromosomes for at least 30 min at–20°C.

!16 Repeat Steps 11–15 three to five times.!17 Store the metaphase chromosomes at

least 24 h in methanol-acetic acid fixa-tive at –20°C.

!18 Do either of the following:" Go to Procedure II." Store metaphase chromosomes in

methanol-acetic acid fixative at –20°Cup to several months.

II. Preparation of chromosomalspreads!1 Pretreat slides as follows:

" Wash slides briefly in a 1:1 mixture of100% ethanol and ether.

" Air dry slides." Wash briefly in redistilled H 2O . (Do

not dry.)!2 Centrifuge metaphase chromosomes from

Procedure I (210 x g) for 10 min at 4°C.!3 Remove and discard all supernatant.!4 Resuspend chromosomal pellet in 1–2 ml

of precooled (–20°C) methanol-aceticacid fixative.

!5 Drop the metaphase chromosomes ontoa moist, pretreated slide from a height ofapproximately 50 cm.

!6 Allow slides to air dry.!7 For optimal metaphase spreads, store

slides at least 5 days in 70% ethanol at4°C.Note: You may store slides up to severalweeks in 70% ethanol at 4°C .

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IVB. For detection of DIG-labeledchromosome-specific probes!1 After hybridization, wash slides in a

Coplin jar according to one of the fol-lowing wash schemes:" 15 min at 40°–48°C with 1 x SSC

containing 50% formamide, pH 7.0;then: 2 x 5 min at room temperaturewith 2 x SSC, pH 7.0or

" 15 min at 37°C with 2 x SSC, pH 7.0.Note: Different probes require differ-ent washes. See Table 1 for the correctwashes to use with each probe.

!2 (O ptional) Incubate slides for 30 min at37°C with 50 ml PBS containing 1%blocking reagent.

!3 Just before use, prepare 100 µl of a 1 µg/mlworking solution of anti-DIG-fluoro-phore conjugate in PBS containing 1%blocking reagent. Note: Each slide to be analyzed requires100 µl of antibody work ing solution. Note: Anti-DIG-fluorophore conjugatecan be Anti-DIG-Fluorescein, Anti-DIG-AMCA, or Anti-DIG-Rhodamine.

!4 Add 100 µl freshly prepared antibodyworking solution to each slide and coverthe complete slide with a coverslip.

!5 Incubate slides for 30 min at 37°C in amoist chamber.

!6 In a Coplin jar, wash slides for 3 x 5 minat room temperature with 2 x SSCcontaining 0.1% Tween® 20.

!7 Counterstain, mount and view slides as for fluorescein-labeled probes (Steps2–7, Procedure IVA).Exception: For experiments using anti-DIG-rhodamine conjugates, use DAPIrather than propidium iodide as acounterstain. The emission wavelengthof propidium iodide is too near that ofrhodamine.Note: Maximum emission wavelengthfor AMCA is 474 nm; for rhodamine,570 nm.

IV. Detection of hybridized sequences

IVA. For detection of fluorescein-labeledchromosome-specific probesNote:Perform Steps 1 and 2 in a Coplin jar.!1 After hybridization, wash slides ac-

cording to one of the following washschemes:" 15 min at 30°–48°C with 1 x SSC

containing 50% formamide, pH 7.0;then: 2 x 5 min at room temperaturewith 2 x SSC, pH 7.0or

" 15 min at 37°C with 2 x SSC, pH 7.0.Note: Different probes require differ-ent washes. See Table 1 for the correctwashes to use with each probe.

!2 For counterstaining, add 50 ml of eitherpropidium iodide solution (100 ng/mlpropidium iodide in PBS) or DAPIsolution (100 ng/ml DAPI in PBS) tothe slides, then incubate the slides for 2–5min in the dark at room temperature.

!3 Wash slides 2–3 min under runningwater.

!4 Air dry the slides in the dark.!5 Apply 20 µl antifade solution [e.g., 2%

DABCO (w/v) in PBS containing 50%glycerol] to each slide.

!6 Add a 24 x 24 mm coverslip.Note: For long term storage of the slides,seal the edges of the coverslip w ith nailpolish and store in the dark at –20°C.

!7 Analyze the slides under a fluorescencemicroscope with the appropriate filters.Note: Maximum emission wavelengthfor fluorescein (FLUO S) is 523 nm; forpropidium iodide, 610 nm; and forDAPI , 470 nm.

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Table 1: Hybridization parametersfor human chromosome-specificsatellite DNA probes. Use the datagiven in this table in Procedures IIIand IV of this article. All probes areavailable from BoehringerMannheim.

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Probe1 for Labeled Formamide (%) in Posthybridization wash conditionschromosomes with hybridization solution Wash 1 (15 min) Wash 2 (2 x 5 min)

1 DIG 2 65% 43°C; 1x SSC + 50% formamide RT; 2 x SSCFLUO S 65% 43°C; 1x SSC + 50% formamide RT; 2 x SSC

2 DIG 65% 48°C; 1x SSC + 50% formamide RT; 2 x SSCFLUO S 65% 48°C; 1x SSC + 50% formamide RT; 2 x SSC

3 DIG 65% 43°C; 1x SSC + 50% formamide RT; 2 x SSC


5 + 1 + 19 DIG 65% 46°C; 1x SSC + 50% formamide RT; 2 x SSCFLUO S 65% 43°C; 1x SSC + 50% formamide RT; 2 x SSC








13 + 21 DIG 65% 43°C; 1x SSC + 50% formamide RT; 2 x SSC

14 + 22 DIG 65% 43°C; 1x SSC + 50% formamide RT; 2 x SSCFLUO S 65% 43°C; 1x SSC + 50% formamide RT; 2 x SSC







X DIG 65% 46°C; 1x SSC + 50% formamide RT; 2 x SSCFLUO S 65% 46°C; 1x SSC + 50% formamide RT; 2 x SSC

Y DIG 50% 37°C; 2 x SSC O mitFLUO S 50% 37°C; 2 x SSC O mit

All human DIG 50% 37°C; 2 x SSC O mitFLUO S 50% 37°C; 2 x SSC O mit

1 The probes all recognize a pattern of alphoid DNA on specific chromosomes, except for the chromosome 1probe (which recognizes satellite III DNA on chromosome 1) and the chromosome Y probe (which recog-nizes a repeat on the long arm of chromosome Y).

2 Abbreviations used: DIG, digoxigenin; FLUOS, 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester; 1xSSC, buffer (pH 7.0) containing 150 mM NaCl and 15 mM sodium citrate; RT, room temperature.

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Results

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Figure 1: FISH of human chromosome-specificsatellite DNA probes to metaphase spreads.Fluorescence signals have been pseudocolored with a CCD camera and a digital imaging system. In Panel a, a DIG-labeled probe specific forchromosomes 5 + 1 + 19 was detected with anti-DIG-rhodamine. Chromosomes were counterstainedwith DAPI. In Panel b, a DIG-labeled probe specificfor chromosome12 wasdetectedwithanti-DIG-fluorescein. Chromosomes were counterstainedwith propidium iodide. Panel c shows the directdetection of chromosome 12 sequences with afluorescein-labeled probe. Chromosomes werecounterstained with propidium iodide.

ReferencesWaye, J. S.; Willard, H. F. (1987) Nucleotidesequence heterogeneity of alpha satellite repetitiveDNA: a survey of alphoid sequences from differenthuman chromosomes. Nucl. Acids Res. 15,7549–7569.Choo, K. H.; Vissel, B.; Nagy, A.; Earle, E.; Kalitsis,P. (1991) A survey of the genomic distribution ofalpha satellite DNA on all the human chromosomes,and derivation of a new consensus sequence. Nucl. Acids Res. 19, 1179–1182.

a

c

b

#$

nent. The fragment length distribution ofthe labeled probes shows a maximum at200–500 bases. 1 µg probe is sufficient for20–50 hybridizations on 24 x 24 mmcoverslips.

These DN A probes are plasmids containinghuman ! satellite DN As. Clones wereisolated from human-hamster hybrid celllines containing the respective humanchromosomes as the only human compo-

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Reagents available from BoehringerMannheim for this procedure

Reagent Description Cat. No. Pack size DNA, MB-grade It prevents unspecific hybridization in all 1467140 500 mg from fish sperm types of membrane or in situ hybridization (50 ml)DAPI Fluorescence dye for staining of 236 276 10 mg

chromosomesAnti-Digoxigenin- Fab Fragments from sheep 1207741 200 µg FluoresceinAnti-Digoxigenin- Fab Fragments from sheep 1207750 200 µg RhodamineAnti-Digoxigenin- Fab Fragments from sheep 1533 878 200 µgAMCA

Reagent Cat. No. Pack size DNA probes, human chromosome" 1 specific, digoxigenin-labeled 1558 765 1 µg (50 µl)" 1 specific, fluorescein-labeled 1558 684 1 µg (50 µl)" 1 + 5 + 19 specific, digoxigenin-labeled 1690 507 1 µg (50 µl)" 1 + 5 + 19 specific, fluorescein-labeled 1690 647 1 µg (50 µl)" 2 specific, digoxigenin-labeled 1666 479 1 µg (50 µl)" 2 specific, fluorescein-labeled 1666 380 1 µg (50 µl)" 3 specific, digoxigenin-labeled 1690 515 1 µg (50 µl)" 4 specific, digoxigenin-labeled 1690 523 1 µg (50 µl)" 6 specific, digoxigenin-labeled 1690 531 1 µg (50 µl)" 7 specific, digoxigenin-labeled 1690 639 1 µg (50 µl)" 7 specific, fluorescein-labeled 1694 375 1 µg (50 µl)" 8 specific, digoxigenin-labeled 1666 487 1 µg (50 µl)" 8 specific, fluorescein-labeled 1666 398 1 µg (50 µl)" 9 specific, digoxigenin-labeled 1690 540 1 µg (50 µl)" 10 specific, digoxigenin-labeled 1690 558 1 µg (50 µl)" 10 specific, fluorescein-labeled 1690 655 1 µg (50 µl)" 11 specific, digoxigenin-labeled 1690 566 1 µg (50 µl)" 11 specific, fluorescein-labeled 1690 663 1 µg (50 µl)" 12 specific, digoxigenin-labeled 1690 574 1 µg (50 µl)" 12 specific, fluorescein-labeled 1690 671 1 µg (50 µl)" 13 + 21 specific, digoxigenin-labeled 1690 582 1 µg (50 µl)" 14 + 22 specific, digoxigenin-labeled 1666 495 1 µg (50 µl)" 14 + 22 specific, fluorescein-labeled 1666 401 1 µg (50 µl)" 15 specific, digoxigenin-labeled 1690 604 1 µg (50 µl)" 16 specific, digoxigenin-labeled 1699 091 1 µg (50 µl)" 17 specific, digoxigenin-labeled 1666 452 1 µg (50 µl)" 17 specific, fluorescein-labeled 1666 371 1 µg (50 µl)" 18 specific, digoxigenin-labeled 1666 509 1 µg (50 µl)" 18 specific, fluorescein-labeled 1666 428 1 µg (50 µl)" 20 specific, digoxigenin-labeled 1666 517 1 µg (50 µl)" 20 specific, fluorescein-labeled 1666 436 1 µg (50 µl)" 22 specific, digoxigenin-labeled 1690 612 1 µg (50 µl)" X specific, digoxigenin-labeled 1666 525 1 µg (50 µl)" X specific, fluorescein-labeled 1666 444 1 µg (50 µl)" Y specific, digoxigenin-labeled 1558 196 1 µg (50 µl)" Y specific, fluorescein-labeled 1558 692 1 µg (50 µl)" Specific for all human chromosomes, digoxigenin-labeled 1558 757 1 µg (50 µl)" Specific for all human chromosomes, fluorescein-labeled 1558 676 1 µg (50 µl)

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metaphase chromosomes with satisfactorymorphology and clear hybridizationsignals (Figure 1).

Adapting the same method and probe tometaphase spreads (Figure 2) allowed us todetermine hybridization efficiency.

ProcedureNote: The DN A probe pUC 1.77 is a cloneof the plasmid vector pUC 9 that containsa 1.77 kb human Eco RI fragment. Theinserted DN A was isolated from humansatellite DN A fraction I I / I I I . This insertcontains mainly a tandemly organizedrepetitive sequence in the region q12 ofchromosome 1 (Cooke and H indley, 1979;Gosden et al., 1981).!1 Label the DN A probe pUC 1.77 with

Digoxigenin-11-dUTP according tostandard nick translation procedures (asdescribed in Chapter 4 of this manual).

!2 Cultivate human lymphocytes fromperipheral blood.

!3 Prepare chromosomes from thelymphocytes by standard techniques.

!4 Resuspend the chromosomes and inter-phase nuclei in methanol/acetic acid(3:1) and store at –20°C.

!5 Perform fluorescence detection withAnti-DIG-Fluorescein, Fab fragmentsas described (Lichter et al., 1990), withthe following modifications:" Incubate for approx. 1 h at 37°C." After the blocking step, do not use

bovine serum albumin in the rest ofthe procedure.

" Modify as necessary to allow FISH insuspension.

!6 After labeling and FISH procedures,pipette 10 µl of the suspension on a slidefor microscopic analysis. Counterstainwith 15 µM propidium iodide (PI) and 5 µM DAPI.

!7 Analyze with an epifluorescence micro-scope (O rthoplan equipped with aPlanapo oil 63 x objective and a 1.40aperture, Leitz, Wetzlar, Germany).

Fluorescence in situ hybridization (FISH )has found widespread application in theanalysis of chromosomes and interphasenuclei fixed on slides (Anastasi et al., 1990;Cremer et al., 1988, 1990; Dekken et al.,1990; Devilee et al., 1988; Kolluri et al.,1990; Lichter et al., 1991; Pinkel et al., 1988;Schardin et al., 1985; Wienberg et al., 1990).

H owever, hybridization of specific DN Aprobes to isolated metaphase chromosomesin suspension offers a new approach tochromosome analysis and chromosomeseparation. Initial investigations were doneon chromosomes obtained from a (Chinesehamster X human) hybrid cell line with bio-tinylated human genomic DN A as theprobe (Dudin et al., 1987, 1988; H ausmannet al., 1991).

So far the technique for FISH in suspensionhas been a modification of FISH tech-niques used for metaphase chromosomesand interphase nuclei fixed on slides. Form-amide (and to some extent dextran sulfate)are obligatory components of this method.

Thus, the technique requires a certain num-ber of washing steps after hybridization.The washing steps for FISH in suspension,however, are based on centrifugal steps.These steps are responsible for a consider-able reduction in the final amount ofchromosomal material.

Additionally, the existing method forFISH in suspension appears to favor anaggregation of the chromosomal materialin suspension.

We report here a hybridization techniquewhich does not need formamide anddextran sulfate. As a model system, we usedthe repetitive specific human DN A probepUC 1.77 (Cooke and H indley, 1979;Emmerich et al., 1989), labeled it withdigoxigenin-11-dUTP by nick-translation,and hybridized it to metaphase chromo-somes in suspension. These chromosomeswere isolated by standard techniques fromhuman lymphocytes.

In preliminary experiments, this techniqueproduced a large number of isolated

Fluorescence in situhybridization of a repetitiveDNA probe to human chromosomes in suspensionD. Celeda1, 2, U . Bettag1, and C . Cremer1

1 Institute for Applied Physics, University of H eidelberg.2 Institute for H uman Genetics and Anthropology, University of H eidelberg, Germany.

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!8 Photograph with, for instance, Fuji-chrome P1600 D film at a final imagemagnification of about 630 x, using Leitzfilter system “I 2/3” for detection ofFITC fluorescence.

Results

Figure 1: FISH of digoxigenin-labeled DNA probepUC 1.77 to isolated human chromosomes insuspension. For details of the hybridizationprocedure, see the text. The sites of hybridizationon the chromosomes appear as yellowish-green“spots” in the centromeric regions. Counterstainingwas with propidium iodide (PI) and DAPI.

Figure 2: FISH of a human metaphase spreadaccording to the same hybridization techniqueused in Figure 1. In this case the pUC 1.77 DNAprobe binds to major hybridization sites (q12 regionof human chromosome 1) that appear as two largeyellowish-green “spots” on two of the largestchromosomes (large arrowheads). Additionally, aminor binding site of the DNA probe (chromosome16; Gosden et al., 1981) is indicated by two minoryellowish-green “spots” on two of the smallerchromosomes (small arrowheads).

"

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Dudin, G.; Steegmayer, E. W.; Vogt, P.; Schnitzer, H.;Diaz, E.; Howell, K. E.; Cremer, T.; Cremer, C. (1988)Sorting of chromosomes by magnetic separation.Human. Genet. 80, 111–116.Emmerich, P.; Loos, P.; Jauch, A.; Hopman, A. H. N.;Wiegant, J.; Higgins, M. J.; White, B. N.; van derPloeg, M.; Cremer, C.; Cremer, T. (1989) Double insitu hybridization in combination with digital imageanalysis: a new approach to study interphasechromosome topography. Exp. Cell Res. 181,126–149.Gosden, J. R.; Lawrie, S. S.; Cooke, H.J. (1981) A cloned repeated DNA sequence in humanchromosome heteromorphisms. Cytogenet. CellGenet. 29, 32–39.Hausmann, M.; Dudin, G.; Aten, J. A.; Heilig, R.;Diaz, E.; Cremer, C. (1991) Slit scan flow cytometryof isolated chromosomes following fluorescencehybridization: an approach of online screening for specific chromosomes and chromosome trans-locations. Z. Naturforsch. 46c, 433–441.Kolluri, R. V.; Manuelidis, L.; Cremer, T.; Sait, S.;Gezer, S.; Raza, A. (1990) Detection of monosomy 7in interphase cells for patients with myeloid dis-orders. Am. J. Hermatol. 33 (2), 117–122.Lichter, P.; Boyle, A. L.; Cremer, T.: Ward, D. C.(1991) Analysis of genes and chromosomes by non-isotopic in situ hybridization. Genet. Anal.Techn. Appl. 8, 24–35.Lichter, P.; Tang, C. C.; Call, K.; Hermanson, G.;Evans, H. J.; Housman, D.; Ward, D. C. (1990) High resolution mapping of human chromosome 11by in situ hybridization with cosmid clones. Science 247, 64–69.Pinkel, D.; Landegent, J.; Collins, C.; Fuscoe, J.;Segraves, R.; Lucas, J.; Gray, J. W. (1988)Fluorescence in situ hybridization with humanchromosome-specific libraries: detection of trisomy21 and translocations of chromosome 4. Proc. Natl.Acad. Sci. USA85, 9138–9142.Schardin, M.; Cremer, T.; Hager, H. D.; Lang, M.(1985) Specific staining of human chromosomes inChinese hamster X human cell lines demonstratesinterphase territories. Hum. Genet. 71, 281–287.Wienberg, J.; Jauch, A.; Stanyon, R.; Cremer, T.(1990) Molecular cytotaxonomy of primates bychromosomal in situ suppression hybridization.Genomics 8, 347–350.

AcknowledgmentsWe thank Prof. Dr. T. Cremer, Intitute ofH uman Genetics and Anthropology,University of H eidelberg, for providing theplasmid pUC 9 and the human lymphocytepreparations. The work was supported bythe Deutsche Forschungsgemeinschaft.

ReferencesAnastasi, J.; Le Beau M. M.; Vardiman, J. W.;Westbrook, C. A. (1990) Detection of numericalchromosomal abnormalities in neoplastichematopoietic cells by in situ hybridization with achromosome-specific probe. Am. J. Pathol. 136,131–139.Cooke, H. J.; Hindley, J. (1979) Cloning of humansatellite III DNA: different components are indifferent chromosomes. Nucleic Acids Res. 10,3177–3197.Cremer, T.; Lichter, P.; Borden, J.; Ward, D. C.;Manuelidis, L. (1988) Detection of chromosomeaberrations in metaphase and interphase tumorcells by in situ hybridization using chromosome-specific library probes. Hum. Genet. 80, 235–246.Cremer, T.; Popp, S.; Emmerich, P.; Lichter, P.;Cremer, C. (1990) Rapid metaphase and interphasedetection of radiation-induced chromosomeaberrations in human lymphocytes by chromosomalsuppression in situ hybridization. Cytometry 11,110–118.Dekken van, H.; Pizzolo, J. G.; Reuter, V. E.;Melamed, M. R. (1990) Cytogenetic analysis ofhuman solid tumors by in situ hybridization with a set of 12 chromosome specific DNA probes.Cytogenet. Cell Genet. 54, 103–107.Devilee, P.; Thierry, R. F.; Kievits, T.; Kolluri, R.;Hopman, A. H. N.; Willard, H. F.; Pearson, P. L.;Cornellisse, C. J. (1988) Detection of chromosomeaneuploidy in interphasenuclei fromhumanprimarybreast tumorsusingchromosome-specific repetitiveDNA probes. Cancer Res. 48, 5825–5830.Dudin, G.; Cremer, T.; Schardin, M.; Hausmann, M.;Bier, F.; Cremer, C. (1987) A method for nucleic acid hybridization to isolated chromosomes insuspension. Hum. Genet. 76, 290–292.

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Reagent Description Cat. No. Pack sizeDIG-Nick Translation 5 x conc. stabilized reaction buffer in 50% 1745 816 160 µlMix* for in situ probes glycerol (v/v)andDNAPolymeraseI, DNaseI,

0.25 mM dATP, 0.25 mM dCTP, 0.25 dGTP, 0.17 mM dTTP and 0.08 mM DIG-11-dUTP

Anti-Digoxigenin- Fab Fragments from sheep 1207741 200 µgFluoresceinDAPI Fluorescence dye for staining of 236 276 10 mg

chromosomes

*This product or the use of thisproduct may be covered by one or more patents of BoehringerMannheim GmbH, including thefollowing: EP patent 0 649 909(application pending).

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5

!7 Incubate 30 min with anti-DIG anti-body at 37°C under a coverslip.

!8 Wash 5 min in PBS.!9 Blot off excess buffer with paper.!10 Finally, mount the chromosomal prepa-

ration in “glycerol-para-phenylenedia-mine-mixture” [1 mg p-phenylenediaminedissolved in 1 ml phosphate bufferedsaline (1 mM sodium phosphate, pH8.0; 15 mM N aCl) containing 50%glycerol].

!11 Inspect with a fluorescence microscopewith the appropriate set of filters.

Comments: Instead of antibodies labeledwith fluorescent dyes, any other antibodyconjugates (enzyme conjugated, gold-labeled) can be used. In the case ofbiotin-labeled DN A, streptavidin can beused instead of antibodies to detect thehybridized DN A. I have tested anumber of these possibilities, and allwork very well (including gold-labeledanti-digoxigenin antibody followed bysilver-enhancement). H owever, in myexperience, the most precise localizationis obtained with immunofluorescence. I tseems to me that the sensitiv ity is alsohigher (or at least the signal/backgroundratio is improved) with fluorescentlylabeled antibodies. Admittedly, thismight be a matter of personal preference.

Results

III. Hybridization and detection!1 Add 5 µl of the hybridization mixture

containing the digoxigenin-labeled probeDN A (from Procedure I) to the chromo-some preparation and cover with acoverslip (18 x 18 mm).

!2 Seal coverslip with rubber cement.!3 Incubate slides at the appropriate

hybridization temperature between50°C and 65°C (depending on the probe,AT-content, sequence homology, etc.)for 4–6 h (for single copy sequences) orfor 1 h (for repetitive sequences).Note: Longer incubations do not mark -edly increase the hybridization signal!

!4 Remove rubber cement, then wash offthe coverslip in 2 x SSC for 2–5 min atroom temperature .

!5 Wash the slide in PBS for 2 min. Removeexcess buffer from the slide by wipingwith soft paper around the chromosomepreparation.Note: Do not let the preparation drycompletely; this produces background.

!6 Apply 5 µl of a 1:10 diluted solution offluorescein- or rhodamine-labeled anti-DIG antibody. The antibodies shouldbe diluted in PBS containing 1 mg/mlbovine serum albumin.

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Figure 1: Double in situ hybridization of a polytene chromosomeof Chironomuswithtwodifferent clones from a chromosomal walk within the sex-determining region ofChironomus piger. The two !-clones contain genomic DNA fragments which are 60 kbpapart. One clone was labeled with digoxigenin and detected with rhodamine-labeled anti-DIGantibody (red); the second clone was labeled with biotin and detected with anti-biotinantibody and fluorescein-labeled secondary antibody (green). The red and green signals canbe seen simultaneously if the fluorescence microscope is equipped with a filter system forsimultaneous blue and green excitation (filter no. 513803, Leica, Wetzlar). For thehybridization, the two probes are mixed 1:1, and the detection is performed with mixedantibody solutions. The method allows a very clear orientation for a chromosomal walk. Wewere able to discriminate the location of two clones which were only approximately 30 kbapart.

"

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99


Reagent Description Cat. No. Pack size DIG-High Prime*, **, *** Complete reaction mixture for random 1585 606 160 µl

primed labeling of DNA with DIG-dUTP (40 reactions)5x solution with: 1 mM dATP, dCTP, dGTP(each), 0.65 mM dTTP, 0.35 mM DIG-11-dUTP, alkali-labile; random primer mixture; 1 unit/µl Klenow enzyme labelinggrade, in reaction buffer, 50% glycerol (v/v)

Biotin-High Prime** Complete reaction mixture for random 1585 649 100 µlprimed labeling of DNA with Biotin-dUTP (25 reactions)5x solution with: 1 mM dATP, dCTP, dGTP(each), 0.65 mM dTTP, 0.35 mM biotin-16-dUTP, random primer mixture,1 unit/µl Klenow enzyme labelinggrade, in reaction buffer, 50% glycerol (v/v)

RNase, DNase-free RNase, DNase-free, can be used directly 1119 915 500 µg (1 ml)without prior heat treatment in any DNAisolation technique

Anti-Digoxigenin- Fab Fragments from sheep 1207 750 200 µgRhodamine


ReferencesLanger-Safer, P. R.; Levine, M.; Ward, D. C. (1982)Immunological method for mapping genes onDrosophila polytene chromosomes. Proc. Natl.Acad. Sci. USA 79, 4381–4385.Schmidt, E. R.; Keyl, H.-G.; Hankeln, T. (1988) In situ localization of two haemoglobin gene clusters in the chromosomes of 13 species ofChironomus. Chromosoma (Berl.) 96, 353–359.

Figure 3: Double in situ hybridization of biotin-and digoxigenin-labeled DNA probes from twodifferent “DNA puffs” in the salivary gland poly-tene chromosome C of Trichosia pubescens(Sciaridae, Diptera). The probes were hybridizedsimultaneously as described in Figure 1. The twosites of hybridization were detected with rhoda-mine-labeled anti-DIG and anti-biotin antibodiesplus fluorescein-labeled secondary antibody. Thetwo differentially labeled sites are so-called “DNApuffs” , which are chromosomal regions with adevelopmentally regulated DNA amplification. Thedouble in situ hybridization method allows veryrapid mapping of the chromosomes, even when the banding structure of the chromosome is notanalyzed in detail.

Figure 2: Double hybridization of a single copyclone from the sex-determining region togetherwith a highly repetitive DNA element. The singlecopy DNA fragment was labeled with digoxigeninand therepetitive element with biotin. The singlecopy DNA(red)hybridizes toasingleband, while therepetitive element (green) produces signals overnumerous chromosomal sites throughout the entirechromosome. Hybridization and detection of the hybridized DNA was essentially the same as in Figure 1.

# #

***EP Patent 0 324 474 granted to and EP Patent application 0 371 262 pending for BoehringerMannheim GmbH.

***This product or the use of thisproduct may be covered by one or more patents of BoehringerMannheim GmbH, including thefollowing: EP patent 0 649 909(application pending).

***Licensed by Institute Pasteur.

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The protocol given here is a derivative ofseveral published methods (Langer-Safer etal., 1982; Schmidt et al., 1988) with someminor modifications that make the methodof in situ hybridization easier, faster, morereliable, and available to anyone who canoperate a microscope.

I. Labeling the hybridization probeFor best labeling results, follow the randomprimed DN A labeling procedure (Proce-dure IA) in Chapter 4 of this manual. Wesuggest a modification of this procedure asdescribed below:!1 Use 0.5–1 µg linearized DN A in 16 µl

redistilled H 2O .!2 Denature in a boiling water bath for

10 min and chill on ice.!3 Add 4 µl DIG-H igh Prime reaction

mix.!4 Mix, centrifuge and incubate either 2 h at

37°C or overnight at room temperature.!5 Stop the labeling procedure by heating

in boiling water for 10 min.!6 Add water and 20 x SSC to give a final

volume of 100 µl with a final concen-tration of 5 x SSC.Note: I f a larger number of prepara-tions is to be hybridized, increase thevolume to 200 µl.

!7 Add 1 volume of 10% SDS to 100 volumesof the mixture in Step 6 (i.e., 1 µl SDS to100 µl of mixture) to give a final concen-tration of 0.1% SDS.

!8 The mixture is ready for hybridizationand can be stored at –20°C for at leastseveral years without any significantdecrease in hybridization efficiency.Comment: In my experience, it isabsolutely unnecessary to removeunincorporated dN TPs from the mixturebefore using the labeled probe. O n thecontrary, purification steps lead to theloss of hybridizable probe DN A and thusto a decrease in hybridization efficiency.

II. Preparation and denaturation ofpolytene chromosomes from Drosophila,Chironomus, or other speciesSquash larval salivary glands in 40% aceticacid according to standard procedure. For long term storage, place slides withsquashed chromosomes in 100% 2-propanolat –20°C.

Prior to the in situ hybridization, denaturethe chromosomal DN A according to thisprocedure:!1 Rehydrate the slides by incubating them,

for 2 min each, in solutions containingdecreasing amounts of ethanol: 70%ethanol, 50% ethanol, 30% ethanol, 0.1 x SSC –2 x SSC.

!2 Digest with RN ase if necessary. Note: This is usually not necessary.

!3 For better preservation of the chromo-somes, include a “heat stabilization” stepby incubating slides in 2 x SSC for atleast 30 min at 80°C.

!4 Incubate the slides for 90 s in 0.1 NN aO H , at room temperature.

!5 Wash slides for 30 s in 2 x SSC.!6 Dehydrate slides by incubating them,

for 2 min each, in solutions containingincreasing amounts of ethanol: 30%ethanol, 50% ethanol, 70% ethanol,95% ethanol.

!7 Air dry the slides for 5 min. Thepreparations are then ready forhybridization.Comment: I f “denatured” chromosomepreparations are to be stored for a longtime, store them in 95% ethanol or 2-propanol at –20°C.

A simplified and efficient protocol for nonradioactivein situhybridization to polytene chromosomes witha DIG-labeled DNA probeProf. Dr. E. R . Schmidt, Institute for Genetics, Johannes Gutenberg-University of Mainz , Germany.

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! Ethanol suspension: Trypsinize cells (ifnecessary), harvest, wash in PBS, fix incold 70% ethanol (–20°C), and droponto glass slides that have been coatedwith poly-L-lysine.

! Slide and coverslip preparations: Growcells on glass slides or coverslips. Fix incold methanol (–20°C) for 5 s, then incold acetone (4°C) for 3 x 5 s. Air drysamples and store at –20°C.Note: Alternatively, use other fixativesfor the slide and coverslip preparations.

! Cytospins: Cytospin floating cells ontoglass slides at 1000 rpm for 5 min. Airdry samples for 1 h at room temperature.Fix and store as with slide and coverslippreparations above.

II. Cell processing"1 Decide whether samples need to be

treated with RN ase. Then do one of thefollowing:! I f the cells need RN ase, go to Step 2.! I f the cells do not need RN ase, go to

Step 3."2 Treat slides with RN ase as follows:

! O verlay each sample with 100 µlRN ase solution (100 µg/ml RN ase Ain 2 x SSC) and a coverslip.

! Incubate cell samples for 1 h at 37°C.! Remove coverslip and wash samples

3 x 5 min with 2 x SSC.! Go to Step 3.

"3 Treat slides with pepsin as follows:! O verlay each sample with 100 µl pep-

sin solution (50–100 µg/ml pepsin in10 mM H Cl) and a coverslip.

! Incubate cell samples for 10–20 minat 37°C.

! Wash samples as follows:– 2 min with 10 mM H Cl– 2 x 5 min with PBS

"4 Post-fix samples as follows:! Incubate samples with PBS contain-

ing 1% (para)formaldehyde for either20 min at 4°C or 10 min at roomtemperature.

! Wash slides 2 x 5 min with PBS.! Dehydrate samples by passing slides

through a series of ethanol solutions(70% , then 96% , then 100% ethanol),incubating 10–60 s in each solution.

Fluorescence in situ hybridization (ISH ) iswidely utilized because of its high sensiti-vity, resolution, and ability to detectmultiple cellular nucleic acid sequences indifferent colors. Fluorescence ISH , however,has disadvantages, such as:! Fluorescence signals fade when they are

exposed to light.! Autofluorescence in, e.g., tissue sections

can interfere with target analysis.

H ere we outline a multicolor enzyme-basedcytochemical detection protocol for nucleicacids in situ. This protocol produces perma-nent cell preparations with non-diffusible,nonfading reaction products. The reactionproducts can be analyzed with brightfield(Speel et al., 1994a), reflection-contrast(Speel et al., 1993), or, in one case (alkalinephosphatase-Fast Red reaction), fluores-cence microscopy (Speel et al., 1992).

We show examples of single- and multiple-target ISH experiments on standard humanlymphocyte metaphase spreads, as well as oninterphase cell preparations. These resultsdemonstrate the potential of this detectionmethodology for metaphase and interphasecytogenetics, e.g., for studying chromosomeaberrations in different cell types (Martini etal., 1995). In addition, this methodology canbe applied in the area of pathology, since theuniversal detection protocol described herecan be combined with other sample prepa-ration procedures, e.g., for tissue sections(H opman et al., 1991, 1992).

The procedures given below are modifi-cations of previously published procedures(Speel et al., 1992, 1993, 1994a, 1994b).

I. Cell preparationsLymphocytes: Prepare chromosomes fromperipheral blood lymphocytes by standardmethods. Fix in methanol:acetic acid (3:1,v/v), and drop chromosomes onto glassslides that have been cleaned with a 1:1 mixof ethanol and ether.

Cultured cells: Make preparations fromcultured normal diploid cells or tumor celllines by one of the following methods:

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Multiple-target DNA in situhybridization withenzyme-based cytochemical detection systemsE. J. M. Speel, F. C . S. Ramaekers, and A . H . N . H opman, Department of MolecularCell Biology & Genetics, University of L imburg, Maastricht, The N etherlands.

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IV. Multiple-target in situhybridization (ISH)

IVA. ISHwithsimultaneousprobeandtargetdenaturation [for probes with unique orhighly repetitive (e.g., centromere probe)sequences]"1 O n each sample, place 10 µl of hybridi-

zation buffer containing labeled probeDN A (prepared as in Procedure III,Step 2).

"2 Cover sample with a 20 x 20 mm cover-slip and (if you wish) seal the coverslipto the slide with rubber cement.

"3 Denature probe and cellular DN Asimultaneously by placing slides at 70°–75°C for 3–5 min on the bottom of ametal box.

"4 Incubate hybridization samples over-night at 37°C.

"5 Go to Procedure V.

IVB. ISH with separate probe and targetdenaturation [for probes with repetitive(e.g. Alu) elements]"1 Incubate the probe mixture (labeled

probe DN A, human or C ot-1 DN A,hybridization buffer; prepared as inProcedure III, Step 2) for 5 min at 75°C.

"2 Chill the denatured probe mixture onice.

"3 Incubate the denatured probe mixturefor 1–4 h at 37°C to pre-anneal therepetitive sequences in the mixture.

"4 Denature the cell samples as follows:! O verlay the cell samples with 70%

formamide in 2 x SSC.! Incubate the slides for 2 min at 70°C

to denature the cells.! Dehydrate the cell samples in a series

of chilled (–20°C) ethanol solutions(70% , then 96% , then 100% ethanol),incubating them for 5 min in eachsolution.

! Air dry the samples."5 O n each denatured cell sample (from

Step 4), place 10 µl denatured, pre-annealed probe mixture (from Step 3).

"6 Incubate hybridization samples over-night at 37°C.

"7 Go to Procedure V.

III. Probe preparation"1 Label the DN A probes (containing either

repetitive or unique sequences) withBiotin-, Digoxigenin-, or Fluorescein-dUTP according to the nick translationprocedure in Chapter 4 of this manual.

"2 Just before use, prepare hybridizationbuffer containing:! 50% or 60% formamide.! 10% dextran sulfate.! 2 x SSC.! 0.2 µg/µl sonicated herring sperm

DN A.! 0.2 µg/µl yeast tRN A.! 1–2 ng labeled probe DN A/µl hybrid-

ization buffer [if the probe containsunique or highly repetitive (e.g.centromere probe) sequences]or2–4 ng labeled probe DN A/µl hybrid-ization buffer, together with an excess(100–1000 fold) of total human DN Aor C ot-1 DN A [if the probe containsrepetitive (e.g.Alu) elements].

"3 Perform ISH by one of the followingmethods:! I f the probe contains unique or highly

repetitive (e.g. centromere probe)sequences, follow procedure IVA.

! I f the probe contains repetitive (e.g.Alu) elements, follow procedure IVB.

! For multiple-target in situ hybridi-zation, prepare DN A probes labeledwith different haptens (biotin, digoxi-genin, or fluorescein), mix themtogether, and follow either ProcedureIVA or Procedure IVB (depending onthe nature of the probes).

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Table 1: Frequently used detectionsystems for enzyme-based in situhybridization.1 Abbreviations used: Ab, antibody;

ABC, avidin-biotinylated enzyme(horseradishperoxidaseor alkalinephosphatase) complex; E, enzyme(horseradishperoxidaseor alkalinephosphatase).

2 Hapten = biotin, digoxigenin, FITC,or DNP.

3 Anti-hapten Ab raised in anotherspecies (e.g., rabbit, goat, swine)can also be used as primary Ab inprobe detection schemes.

"6 Repeat Step 5 with the next incubationin the detection system (Table 1) until allincubations are complete.

"7 After all incubations in the detection sys-tem are complete, wash samples 5 minwith PBS.

"8 Visualize according to one of the proce-dures in Section VII.

"9 If the detection procedure uses the sameenzyme (peroxidase or alkaline phos-phatase) to detect two different probes,do the following:! Inactivate the enzyme on the first

detecting molecule by incubating thesample for 10 min at room temper-ature with 10 mM H Cl.

! Repeat Steps 5–8 with a seconddetection system that recognizes thesec-ond probe (Speel et al., 1994b).

V. Post-hybridization washes"1 Perform the following stringent washes

of the samples (from either ProcedureIVA or Procedure IVB) at 42°C:! 2 x 5 min with 2 x SSC containing

50% (or 60% ) formamide and 0.05%Tween® 20.

! 2 x 5 min with 2 x SSC."2 Depending upon the nature of the

probe, do one of the following:! I f the probe contains repetitive (e.g.

Alu) elements, wash the samples 2 x5 min at 60°C with 0.01 x SSC.

! I f the probe does not contain repeti-tive elements, skip this step.

VI. Enzyme-based cytochemical detection

VIA. Single color detection

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Probe Incubationlabel 1 2 3

Biotin Avidin-E1

Biotin Avidin-E Biotinylated anti-avidin Ab Avidin-EH apten2 Anti-hapten Ab-EH apten Mouse3 anti-hapten Ab Anti-mouse Ab-EH apten Mouse anti-hapten Ab Rabbit anti-mouse Ab-E Anti-rabbit Ab-EH apten Mouse anti-hapten Ab Biotinylated anti-mouse Ab ABCH apten Mouse anti-hapten Ab DIG-labeled anti-mouse Ab Anti-DIG Ab-E

"1 Wash samples briefly with 4 x SSCcontaining 0.05% Tween® 20.

"2 Incubate samples for 10 min at 37°Cwith 4 x SSC containing 5% nonfat drymilk.

"3 Choose an appropriate enzyme-baseddetection system (Table 1).

"4 Dilute detecting molecules as follows:! Dilute avidin conjugates in 4 x SSC

containing 5% nonfat dry milk.! Dilute antibody conjugates in PBS

containing 2–5% normal serum and0.05% Tween® 20.

"5 For the first incubation in the detectionsystem (Table 1), do the following:! Incubate samples with diluted detect-

ing molecule for 30 min at 37°C.! Wash samples 2 x 5 min in the appro-

priate wash buffer (4 x SSC for avidin;PBS for antibodies) containing 0.05%Tween® 20.

VIB. Multiple target, multicolor detectionTo detect multiple probes labeled withdifferent haptens, use a combination ofdetection systems. For example, Table 2outlines a protocol for triple-target in situhybridization. For protocols with double-target in situ hybridizations, see H opmanet al. (1986), Emmerich et al. (1989),Mullink et al. (1989), H errington et al.(1989), Kerstens et al. (1994), and Speel etal. (1995).

Note: Alternatively, if two peroxidase orphosphatase reactions are used in thedetection protocol (Speel et al., 1994b),inactivate the enzyme after the firstdetection reaction in 10 mM H Cl for 10min at room temperature, then performthe second detection reaction (as inProcedure VIA above).

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VIIB. Horseradish peroxidase-tetramethylbenzidine (PO-TMB)"1 Dissolve 100 mg sodium tungstate

(Sigma) in 7.5 ml 100 mM citrate-phosphate buffer (pH 5.1). Adjust thepH of the tungstate solution to pH5.0–5.5 with 37% H Cl.

"2 Just before use, dissolve 20 mg dioctylsodium sulfosuccinate (Sigma) and 6 mg3,3' ,5,5' -tetramethylbenzidine (TMB,Sigma) in 2.5 ml 100% ethanol at 80°C.

"3 Prepare 10 ml color reagent by combin-ing the tungstate solution (Step 1), theTMB solution (Step 2), and 10 µl 30%H 2O 2.

"4 O verlay each sample with 100 µl colorreagent and a coverslip.

"5 Incubate samples for 1–2 min at 37°C."6 Wash samples 3 x 1 min with ice-cold

100 mM phosphate buffer (pH 6.0) and(if you wish) dehydrate them.

"7 Coverslip with an organic mountingmedium or immersion oil.

VII. Visualization

Detection step Incubation Incubationtime2 temperature

1. Detect biotin with AvPO 1 (diluted 1:50) 20 min 37°C

2. Visualize PO by Procedure VIIA 5 min 37°C(PO -DAB, brown signal)

3. Inactivate residual AvPO with 10 mM H Cl. 10 min RT

4. Detect digoxigenin and FITC with MADIG 30 min 37°Cand RAFITC (each diluted 1:2000)

5. Detect anti-digoxigenin and anti-FITC with 30 min 37°CGAMAPase (diluted 1:25) and SWARPO (diluted 1:100)

6. Visualize APase activity by Procedure VIIC 5–10 min 37°C(APase-Fast Red, red signal)

7. Visualize PO activity by Procedure VIIB 1–2 min 37°C(PO -TMB, green signal)

8. Counterstain with hematoxylin 1 sec RT

9. Air dry 10 min RT

10. Embed in a protein matrix3 10 min 37°C

Depending upon the type of microscopyto be used for analysis (Table 3) and thedetecting molecule, choose one of thefollowing procedures to visualize thehybrids. In our hands, each of these proce-dures is optimal for in situ hybridization.

VIIA. Horseradish peroxidase-diaminobenzidine (PO-DAB)"1 Mix color reagent just before use:

! 1 ml 3,3-diaminobenzidine tetra-chloride (DAB; Sigma) stock (5 mgDAB/ml PBS).

! 9 ml PBS containing 0.1 M imidazole,pH 7.6.

! 10 µl 30% H 2O 2."2 O verlay each sample with 100 µl color

reagent and a coverslip."3 Incubate samples for 5–15 min at 37°C."4 Wash samples 3 x 5 min with PBS and (if

you wish) dehydrate them."5 Coverslip with an aqueous or organic

mounting medium.

Enzyme label Substrate Precipitate colors in microscopyBrightfield Reflection-contrast Fluorescence

PO 2 H 2O 2/DAB Brown White –H 2O 2/TMB Green Pink/Red –

APase N -ASMX-P/FR Red Yellow RedBCIP/N BT Purple Yellow/O range –

Table 2: Detection protocol for triple-target in situ hybridizationwith a biotin-, digoxigenin-, and aFITC-labeled probe.1 Abbreviations used: Ab, antibody;

APase, alkaline phosphatase;AvPO, PO-conjugated avidin(Vector); DAB, diaminobenzidine; GAMAPase, APase-conjugatedgoat anti-mouse Ab (DAKO);MADIG, mouse anti-digoxigeninAb; PO, horseradish peroxidase;RAFITC, rabbit anti-FITC Ab(DAKO); RT, room temperature;SWARPO, PO-conjugated swineanti-rabbit Ab (DAKO).

2 For details of detection reactions,see Procedure VIA. For details ofvisualization reactions, seeProcedures VIIA–VIID.

3 For details of protein matrix, seeProcedure VIII.

Table 3: Enzyme reactionprotocols and colors in differenttypes of light microscopy1.1 Other enzyme reactions that have

been used for ISH are described inSpeel et al. (1993, 1995).

2 Abbreviationsused: APase, alka-line phosphatase; BCIP, bromo-chloro-indolyl phosphate; DAB,diaminobenzidine; FR, Fast RedTR; N-ASMX-P, naphthol-ASMX-phosphate; NBT, nitroblue tetra-zolium; PO, horseradish peroxi-dase; TMB, tetramethylbenzidine.

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ResultsVIIC. Alkaline phosphatase-Fast Red(APase-Fast Red)"1 Mix color reagent just before use:

! 4 ml TM buffer [200 mM Tris-H Cl(pH 8.5), 10 mM MgCl2] containing5% polyvinyl alcohol (PVA, MW40,000; Sigma).

! 250 µl TM buffer containing 1 mgnaphthol-ASMX-phosphate (Sigma).

! 750 µl TM buffer containing 5 mgFast Red TR salt (Sigma).


"3 Incubate samples for 5–15 min at 37°C."4 Wash samples 3 x 5 min with PBS."5 Coverslip with an aqueous mounting

medium.

VIID. Alkaline phosphatase-bromochloro-indolyl phosphate (APase-BCIP/NBT)Follow the standard procedure given inChapter 2 of this manual.

VIII. Embedding and light microscopy"1 Prepare samples for microscopy by

doing either of the following:! I f samples require a single mounting

medium, embed stained samples asdescribed in Procedure VII.

! I f multiple precipitation reactionswould require different (aqueous ororganic) embedding mediums, applyinstead a protein embedding layer bysmearing 50 µl of a 1:1 mixture ofBSA solution (40 mg/ml in deionizedH 2O ) and 4% formaldehyde onto theslides. Air dry for 10 min at 37°C.Note: This protein embedding layercan be used to prevent solubilizationof enzyme precipitates during alltypes of light m icroscopy (Speel et al.,1993, 1994a).

"2 Perform brightfield, fluorescence andreflection contrast microscopy asdescribed in the literature (Speel et al.,1992, 1993, 1994b; Cornelese-Ten Velde etal., 1989).

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Figure 1: Single-target ISH on a normal humanlymphocytemetaphasespreadwitha peroxidasedetection system. The probe was a biotinylatedcosmid that recognizes 40 kb of chromosome11q23. The detection system included monoclonalanti-biotin Ab (DAKO), rabbit anti-mouseAb-PO,and thePO-DABreaction. The sample wascounterstained with hematoxylin and viewed bybrightfield microscopy.

Figure 2: Double-target ISH on normal humanumbilical vein endothelial cells with brightfieldviewing. The centromere of chromosome 1 (brown)was detected with a biotinylated probe; that ofchromosome 7 (red), with a digoxigenin-labeledprobe. The detection steps included (1) avidin-PO,(2) monoclonal anti-digoxigenin Ab and rabbit anti-mouse Ab-APase, (3) the APase-Fast Red reaction,and (4) the PO-DAB reaction. The sample wascounterstained with hematoxylin and viewed bybrightfield microscopy.

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Figure4:Triple-target ISHona normal humanlymphocyte metaphase spread. Probes specificfor the centromeres of chromosomes 1 (brown), 7 (red), and 17 (green) were labeled with biotin,digoxigenin, and FITC, respectively. Detection,counterstaining, and embedding in a BSA proteinlayer was as outlined in the text (Table 2). Thesample was viewed by brightfield microscopy.

Figure 5: Triple-target ISH on human bladdertumor cell line T24. The probes and experimentalprocedures were the same as in Figure 4.

Reprinted from Speel, E. J. M.; Kamps, M.; Bonnet, J.;Ramaekers, F. C. S.; Hopman, A. H. N.; (1993) Multicolorpreparations for in situ hybridization using precipitating enzyme cytochemistry in combination with reflectioncontrast microscopy. Histochemistry 100, 357–366 withpermission of Springer-Verlag GmbH & Co. KG.

Figure 6: Double-target ISH on a normal humanlymphocyte metaphase spread with reflectioncontrast viewing. Thecentromereof chromosome1(yellow)wasdetectedwithabiotinylated probe; that of chromosome 17 (white), with a digoxigenin-labeled probe. The detection steps included (1)monoclonal anti-biotin Ab and rabbit anti-digoxi-geninAb, (2)horse anti-mouse Ab-biotin andswineanti-rabbit Ab-PO, (3)streptavidin-biotinylatedAPase complex, (4) the APase-Fast Red reaction,and (5) the PO-DAB reaction (Speel et al., 1993).The sample was not counterstained, but wasembedded in a BSA protein layer and viewed byreflection contrast microscopy.

Figure 3: Same ISH experiment as in Figure 1,but with a fluorescent alkaline phosphatasedetection system. The detection system for thebiotinylatedcosmidprobe includedmonoclonal anti-biotin, horse anti-mouse Ab-biotin, avidin-biotinylated APase complex, and the APase-FastRed reaction (Speel et al., 1994a). The sample wascounterstained with Thiazole Orange (MolecularProbes) and viewed by fluorescence microscopy.

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Martini. E.; Speel, E. J. M.; Geraedts, J. P. M.;Ramaekers, F. C. S.; Hopman, A. H. N. (1995)Application of different in situ hybridizationdetection methods for human sperm analysis. Hum. Reprod. 10, 855–861.Mullink, H.; Walboomers, J. M. M.; Raap, A. K.;Meyer, C. J. L. M. (1989) Two colour DNA in situhybridization for the detection of two viral genomesusing non-radioactive probes. Histochemistry 91,195–198.Speel, E. J. M.; Schutte, B.; Wiegant, J.; Ramaekers,F. C. S.; Hopman, A. H. N. (1992) A novel fluores-cence detection method for in situ hybridization,based on the alkaline phosphatase-Fast Redreaction. J. Histochem. Cytochem. 40, 1299–1308.Speel, E. J. M.; Kamps, M.; Bonnet, J.; Ramaekers,F. C. S.; Hopman, A. H. N. (1993) Multicolourpreparations for in situ hybridization usingprecipitating enzyme cytochemistry in combinationwith reflection contrast microscopy. Histochemistry100, 357–366.Speel, E. J. M.; Herbergs, J.; Ramaekers, F. C. S.;Hopman, A. H. N. (1994a) Combined immunocyto-chemistry and fluorescence in situ hybridization for simultaneous tricolor detection of cell cycle,genomic, and phenotypic parameters of tumor cells.J. Histochem. Cytochem. 42, 961–966.Speel, E. J. M.; Jansen, M. P. H. M.; Ramaekers, F. C. S.; Hopman, A. H. N. (1994b) A novel triple-color detection procedure for brightfield micros-copy, combining in situ hybridization with immuo-cytochemistry. J. Histochem. Cytochem. 42,1299–1307.Speel, E. J. M.; Ramaekers, F. C. S.; Hopman, A. H. N.(1995) Detection systems for in situ hybridization, andthe combination with immunocytochemistry.Histochem. J., in press.

ReferencesCornelese-Ten Velde, I.; Wiegant, J.; Tanke, H. J.;Ploem, J. S. (1989) Improved detection andquantification of the (immuno)peroxidase productusing reflectioncontrast microscopy. Histochemistry92, 153–160.Emmerich, P.; Loos, P.; Jauch, A.; Hopman, A. H. N.;Wiegant, J.; Higgins, M. J.; White, B. N.; Van derPloeg, M.; Cremer, C.; Cremer, T. (1989) Double in situ hybridization in combination with digitalimage analysis: a new approach to study interphasechromosome topography. Exp. Cell Res. 181,126–140.Herrington, C. S.; Burns, J.; Graham, A. K.; Bhatt, B.; McGee, J. O’D. (1989) Interphasecyto-genetics using biotin and digoxygenin labeledprobes II: simultaneous differential detection ofhuman and papilloma virus nucleic acids in individual nuclei. J. Clin. Pathol. 42, 601–606.Hopman, A. H. N.; Wiegant, J.; Raap, A. K.;Landegent, J. E.; Van der Ploeg, M.; Van Duijn, P.(1986) Bi-color detection of two target DNAs by non-radioactive in situ hybridization.Histochemistry 85, 1–4.Hopman, A. H. N.; Van Hooren, E.; Van de Kaa, C. A.;Vooijs, G. P.; Ramaekers, F. C. S. (1991) Detection of numerical chromosomeaberrations using in situhybridization in paraffin sections of routinelyprocessed bladder cancers. Modern Pathol. 4,503–513.Hopman, A. H. N.; Poddighe, P. J.; Moesker, O.;Ramaekers, F. C. S. (1992) Interphase cytogenetics:an approach to the detection of genetic aberrationsin tumours. In: McGee, J. O’D.; Herrington, C. S.(Eds.) Diagnostic Molecular Pathology, A PracticalApproach, 1st ed. New York: IRL Press, 141–167.Kerstens, H. M. J.; Poddighe, P. J.; Hanselaar, A. G. J. M. (1994) Double-target in situ hybridizationin brightfield microscopy. J. Histochem. Cytochem.42, 1071–1077.

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Reagent Description Cat. No. Pack size RNase A Dry powder 109 142 25 mg

109 169 100 mgPepsin Aspartic endopeptidase with broad 108 057 1 g

specificity 1693 387 5 gDigoxigenin-11-dUTP, Tetralithium salt, 1 mM solution 1093 088 25 nmolalkali-stable (25 µl)

1558 706 125 nmol(125 µl)

1570 013 5x125 nmol(5x125 µl)

Fluorescein-12-dUTP Tetralithium salt, 1 mM solution 1373 242 25 nmol(25 µl)

Tetramethyl- Tetralithium salt, 1 mM solution 1534 378 25 nmolrhodamine-6-dUTP (25 µl)AMCA-6-dUTP Tetralithium salt, 1 mM solution 1534 386 25 nmol

(25 µl)Biotin-16-dUTP Tetralithium salt, 1 mM solution 1093 070 50 nmol

(50 µl)DNase I Lyophilizate 104 159 100 mgDNA Polymerase I Nick Translation Grade 104 485 500 units

104 493 1000 unitstRNA From baker’s yeast 109 495 100 mg

109 509 500 mgDNA, Cot-1, human Solution in 10 mM Tris-HCl, 1 mM EDTA, 1581 074 500 µg

pH 7.4, Cot-1 DNA is used in chromosomal (500 µl)in situ suppression (CISS).

Tween® 20 1332 465 5x10 mlAnti-Digoxigenin Clone 1.71.256, mouse IgG 1, ! 1333 062 100 µgAnti-Mouse Ig- F(ab)2 fragment 1214 624 200 µgDigoxigeninAnti-Digoxigenin-POD Fab fragments from sheep 1207 733 150 UAnti-Digoxigenin-AP Fab fragments from sheep 1093 274 150 UNBT/BCIP, Solution of 18.75 mg/ml nitro-blue 1681 451 8 ml(stock solution) tetrazolium chloride and 9.4 mg/ml

5-bromo-4-chloro-3-indolyl phosphate,toluidine-salt in 67% DMSO (v/v)

Tris Powder 127 434 100 g708 968 500 g708 976 1 kg

BSA Highest quality, lyophilizate 238 031 1 g238 040 10 g

! Prepare Fast Red TR stock solutionby dissolving 5 mg Fast Red TR(vial 2) in 200 µl distilled H 2O .

! Mix 10 µl H N PP stock solution (pre-mixed, vial1),10 ml Fast Red TR stocksolution, and 1 ml detection buffer[100 mM Tris-H Cl, 100 mM N aCl, 10 mM MgCl2; pH 8.0 (20°C)].

! Filter the H N PP/Fast Red TR mixthrough a 0.2 µm nylon filter.Caution: The Fast Red TR stocksolution should be no more than 4weeks old. The H N PP/Fast Red TRmix should be no more than a fewdays old. A lways filter the mix justbefore use.

"6 Cover each sample with 100 µl filteredH N PP/Fast Red TR mix and a cover-slip, then incubate slides for 30 min atroom temperature.

"7 Wash the slides 1x at room temperaturewith washing buffer [100 mM Tris-H Cl,150 mM N aCl, 0.05% Tween® 20; pH7.5 (20°C)].

"8 Repeat Steps 6 and 7 three times.Note: I f you are detecting abundant ormultiple copy sequences, sk ip Step 8.O ne incubation is enough.

"9 Wash the slides for 10 min with distilledH 2O .

III. Fluorescence microscopy"1 In a Coplin jar, counterstain slides with

50 ml DAPI solution (100 ng DAPI/mlPBS) for 5 min in the dark at roomtemperature.

"2 Wash slides under running water for 2–3 min.

"3 Air dry slides in the dark."4 Add 20 µl DABCO antifading solution

(PBS containing 50% glycerol and 2%DABCO ) to each slide.

"5 Cover each slide with a 24 x 24 mmcoverslip.Note: For long term storage of the slides,seal the edges of the coverslip w ith nailpolish and store in the dark at –20°C.

"6 View the slides by fluorescence micros-copy, using appropriate filters.

We describe here a high sensitivity indirectdetection procedure for DIG-labeledhybridization probes. The procedure usesthe components of the H N PP FluorescentDetection Set to form a fluorescent precip-itate of H N PP (2-hydroxy-3-naphthoicacid-2'-phenylanilide phosphate) and FastRed TR at the site of hybridization.

This procedure can be used to detect singlecopy sequences as small as 1 kb on humanmetaphase chromosomes.

I. In situ hybridization with DIG-labeled probesPrepare chromosomal spreads, label hybridization probes with digoxigenin(DIG), hybridize probes to chromosomes,and perform posthybridization washes ofthe sample according to standard proce-dures, e.g. those listed in Chapters 4 and 5of this manual.

II. Detection of DIG-labeled probes"1 Prepare a fresh 1:500 dilution of alkaline

phosphatase-conjugated sheep anti-DIGantibody in blocking buffer [100 mMTris-H Cl, 150 mM N aCl, 0.5% blockingreagent; pH 7.5 (20°C)].Note: Do not store the diluted antibodyconjugate more than 12 h at 4°C .

"2 Pipette 100 µl of diluted anti-DIGantibody conjugate onto each slide andadd a coverslip.

"3 Incubate slides for 1 h at 37°C in a moistchamber.

"4 Wash the slides at room temperature asfollows:! 3 x10 min with washing buffer

[100 mM Tris-H Cl, 150 mM N aCl,0.05% Tween® 20; pH 7.5 (20°C)].

! 2 x10 min with detection buffer [100 mM Tris-H Cl,100 mM N aCl, 10 mM MgCl2; pH 8.0 (20°C)].

"5 Prepare fresh H N PP/Fast Red TR mixas follows:Note: N umbered v ials are componentsof the H N PP Fluorescent Detection Set.

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DNA in situhybridization with an alkaline phosphatase-based fluorescent detection systemDr. G . Sagner, Research Laboratories, Boehringer Mannheim GmbH , Penzberg, Germany.

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Note: The maximum emission wave-length of dephosphorylated H N PP/FastRed TR is 562 nm. H owever, since theemission peak is broad (540–590 nm),you may use the filter sets for eitherfluorescein or rhodamine to analyze theH N PP/Fast Red TR product.

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Figure 1: Comparison of fluorescein and HNPP signal intensity. A DIG-labeled paintingprobe specific for human chromosome 1 was hybridized to metaphase chromosomalspreads. The DIG-labeled probe was detected with (Panel a)anti-DIG-fluoresceinconjugateor(Panel b)anti-DIG-alkaline phosphate conjugateand theHNPP/Fast Reddetection reactiondescribed in thetext. Thefluoresceinsignal (Panel a) required a 15 s exposurewhile theHNPP/Fast Redsignal (Panel b) required a 0.2 s exposure. Fluorescent images were pseudo-colored with a CCD camera and a digital imaging system. Chromosomes were counter-stained with DAPI.

Figure 2: HNPP/Fast Redfluorescence viewed with a filterfor DAPI. A DIG-labeled paintingprobe specific for humanchromosome 1 was hybridized tometaphase chromosomal spreads,then detected with anti-DIG-alkalinephosphate conjugate and theHNPP/Fast Reddetection reactionasinFigure1. The HNPP/Fast Redsignal was viewed under a filter thatwas optimal for DAPI (emissionmaximum, 470 nm), but not forHNPP (emission maximum, 562 nm).The fluorescence was photographeddirectly without digital manipulation.Chromosomes were counterstainedwith DAPI.

a

b

ResultsThe fluorescent signal obtained with theH N PP/Fast Red procedure describedabove is more intense than a direct signalfrom fluorescein. In a comparison experi-ment (Figure 1), the H N PP/Fast Redreaction product was visible after a 70-foldshorter exposure (0.2 sec exposure vs. 15 sexposure) than the fluorescein signal. Evenwhen the H N PP/Fast Red signal is viewedunder a less than optimal filter (Figure 2),the fluorescence is clearly visible.

Reagent Description Cat. No. Pack size Tris Powder 127 434 100 g

708 968 500 g708 976 1 kg

Blocking Reagent Powder 1096 176 50 gAnti-Digoxigenin-AP Fab Fragments from sheep 1093 274 150 UTween® 20 1332 465 5x10 mlHNPP Fluorescent For sensitive fluorescent detection of non- 1758 888 Set for 500Detection Set radioactively labeled nucleic acids in FISH reactions

fluorescence in situ hybridization (FISH)and membrane hybridization

DAPI Fluorescence dye for staining of 236 276 10 mgchromosomes


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Fluorochromes have been used mainly onacetone-fixed cell preparations, since thematerial can be mildly post-fixed (usuallywith paraformaldehyde) after antigendetection and used directly for fluores-cence ISH without any further pre-treatment (Weber-Matthiesen et al., 1993).H owever, amplification steps for both ICCand ISH signals are often necessary forclear visualization. In such cases, enzy-matic ISH pre-treatment after ICC lowersfluorescent ICC staining dramatically(Speel et al., 1994a).

Enzyme precipitation reactions have alsobeen used efficiently for combined ICC/ISH staining of proteins in cell prepara-tions and tissue sections. Enzyme precip-itation products that withstand theproteolytic digestion and denaturationsteps used in the ISH procedure include:

! Several precipitates (Fast Red, N ewFuchsin, and BCIP/N BT) formed byalkaline phosphatase

! The diaminobenzidine precipitate formedby horseradish peroxidase

! The BCIG (X-Gal) precipitate formedby !-galactosidase

In these cases, the digestion and denatur-ation steps of the ISH procedure removethe antibody and enzyme detection layers,but the precipitate remains firmly in place.The stability of the ICC precipitate thusprevents unwanted cross-reaction betweenthe detection procedures for ISH and ICC.

H ere we present a combined ICC/ISHprocedure which describes compatibledetection systems for fluorescence orbrightfield microscopy (Table 1). Addi-tionally, this procedure allows thelocalization of incorporated BrdU byfluorescence microscopy.

The combination of in situ hybridization(ISH ) and immunocytochemistry (ICC)enables us, e.g., to simultaneously demon-strate mRN A and its protein product inthe same cell, to immunophenotype cellscontaining a specific chromosomal aber-ration or viral infection, or to characterizecytokinetic parameters of tumor cell popu-lations that are genetically or pheno-typically aberrant. The factors thatdetermine the success and sensitivity of acombination ICC and nonradioactive ISHprocedure include:

! Preservation of cell morphology andprotein epitopes

! Accessibility of nucleic acid targets! Lack of cross-reaction between the

different detection procedures! Good color contrast! Stability of enzyme cytochemical precip-

itates and fluorochromes

Since several steps in the ISH procedure(enzymatic digestion, post-fixation, denat-uration at high temperatures, and hybridi-zation in formamide) may destroy antigenicdeterminants, ICC usually precedes ISH ina combination procedure. A variety of suchcombined ICC/ISH procedures have beenreported with either enzyme precipitationreactions (Mullink et al., 1989; Van denBrink et al., 1990; Knuutila et al., 1994;Speel et al., 1994b), fluorochromes (Vanden Berg et al., 1991; Weber-Matthiesen etal., 1993), or a combination of both (Strehl& Ambros, 1993; Zheng et al., 1993;H erbergs et al., 1994; Speel et al., 1994a).The procedures can be subdivided into twogroups, those which use fluorochromes forICC, and those which use enzyme reactions.

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Procedures for In Situ H ybridization to Chromosomes, Cells, and Tissue SectionsISH to cells

Combined DNA in situhybridization andimmunocytochemistry for the simultaneousdetection of nucleic acid sequences, proteins, andincorporated BrdU in cell preparationsE. J. M. Speel, F. C . S. Ramaekers, and A . H . N . H opman, Department of MolecularCell Biology & Genetics, University of L imburg, Maastricht, The N etherlands.

1 Amplification steps may benecessary for detection of lowamounts of antigen, nucleic acidtarget, or incorporated BrdU.

2 Abbreviations used: Ab, antibody;AMCA, aminomethylcoumarinacetic acid; APase, alkalinephosphatase; BCIG,bromochloroindolyl-galactoside;BrdU, bromodeoxyuridine; DAB,diaminobenzidine; DIG,digoxigenin; FITC, fluoresceinisothiocyanate; !-Gal, !-galactosidase; ICC,immunocytochemistry; ISH, in situhybridization; PO, peroxidase;TMB, tetramethylbenzidine

3 The fluorochrome used in the BrdUvisualization step should be differ-ent from the one used in the ISHvisualization step.

"4 Incubate slides for 45 min at roomtemperature with an appropriate second-ary antibody conjugate. Use thefollowing to decide which antibodyconjugate to use:! I f you wish to detect the ICC antigen,

the ISH antigen, and (optionally)BrdU labeling by fluorescence micros-copy, use a secondary antibody that isconjugated to alkaline phosphatase(APase).

! I f you wish to detect the ICC antigenand the ISH antigen under brightfieldmicroscopy, use a secondary antibodythat is conjugated to !-galactosidase(!-Gal).

"5 Wash slides as follows:! 5 min with PBS containing 0.05%

Tween® 20.! 5 min with PBS.

Note: For amplification of the ICCsignal, you may add a third antibodystep after this wash. For details ofpossible antibodies to use in an ampli-fied three-antibody detection procedure,see Table 1 of the article “Multipletarget DN A in situ hybridization withenzyme-based cytochemical detectionsystems” on page 102 of this manual.

"6 Visualize the antibody-antigen com-plexes according to either ProcedureIIIA (for APase conjugates) or Proce-dure IIIB (for !-Gal conjugates).

The procedures given below aremodifications of previously publishedprocedures (Speel et al., 1994a, 1994b).

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Table1:Detectionsystemsfor combinedICC/ISH1.

For analysis byFluorescence microscopy Brightfield microscopy

ICC2 Detection by Anti-antigen Ab-APase Anti-antigen-!-gal

Visualization by APase-Fast Red !-Gal-BCIG

ISH Probe FITC- or AMCA-labeled DIG- or biotin-labelednucleic acid nucleic acid

Detection and Direct viewing PO - or APase-labeled anti-visualization by DIG Ab or anti-biotin Ab

plusPO -DAB, PO -TMB, orAPase-Fast Red reaction

BrdU Detection by AMCA- or FITC- –labeling labeled3 anti-BrdU Ab

Visualization by Direct viewing –

I. Cell preparations and BrdU labeling"1 (O ptional) To label cells, add BrdU

(final concentration, 10 µM) to theculture medium 30 min beforeharvesting the cells (Speel et al., 1994a).

"2 Prepare cultured normal diploid cells ortumor cell lines (labeled or unlabeled)by one of the following methods:! Slide and coverslip preparations: Grow

cells on glass slides or coverslips. Fix incold methanol (–20°C) for 5 s, then incold acetone (4°C) for 3 x 5 s. Air drysamples and store at –20°C.Note: Alternatively, use other fixativesfor the slide and coverslip preparations.

! Cytospins: Cytospin floating cellsonto glass slides at 1000 rpm for 5 min.Air dry samples for 1 h at roomtemperature. Fix and store as withslide and coverslip preparations above.

II. Detection of antigen by immunocytochemistry (ICC) "1 Incubate slides for 10 min at room

temperature with PBS-Tween-N GS (PBSbuffer containing 0.05% Tween® 20 and2–5% normal goat serum).

"2 Incubate slides for 45 min at room tem-perature with an appropriate dilution ofantigen-specific primary antibody inPBS-Tween-N GS.

"3 Wash slides for 2 x 5 min with PBScontaining 0.05% Tween® 20.

#

IV. Cell processing for in situ hybridization"1 Wash slides for 2 min at 37°C with

10 mM H Cl."2 Digest samples with pepsin as follows:

! O verlay each sample with pepsinsolution (100 µg/ml pepsin in 10 mMH Cl).

! Incubate cell samples for 10–20 minat 37°C.

"3 Wash samples for 2 min at 37°C with 10 mM H Cl.Note: For cells stained with the !-Gal-BCIG reaction, you may (if you wish)dehydrate the slides after washing them.

"4 Post-fix samples for 20 min at 4°C withPBS containing 1% paraformaldehyde.

"5 Wash samples as follows:! 5 min with PBS.! 5 min with 2 x SSC.

V. In situ hybridization (ISH)"1 Perform a standard ISH detection and

visualization procedure on the slides asdescribed elsewhere in this manual. Useeither:! Fluorescence-based procedures (FITC,

AMCA) (when APase-conjugatedantibodies were used for ICC).

! Enzyme-based procedures (PO -DAB,PO -TMB, APase-Fast Red) (when !-Gal-conjugated antibodies wereused for ICC).

Example: For a detailed description of enzyme-based ISH procedures, see“Multiple target DN A in situ hybridi-zation w ith enzyme-based cytochem -ical detection systems” on page 100 ofthis manual.

"2 Include appropriate controls in the ISHprocedure to ensure the lack of cross-reaction between ICC and ISH .Note:The ICC precipitate remains firmlyin place during ISH . The stability of the ICC precipitate usually preventsunwanted cross-reaction between thedetection procedures for ISH and ICC.

III. Visualization of ICC antigen

IIIA. APase-Fast Redreaction(for producinga red precipitate visible under either fluorescence or brightfield microscopy)"1 Mix color reagent just before use:

! 4 ml TM buffer [200 mM Tris-H Cl(pH 8.5), 10 mM MgCl2] containing5% polyvinyl alcohol (PVA, MW40,000; Sigma).

! 250 µl TM buffer containing 1 mgnaphthol-ASMX-phosphate (Sigma).

! 750 µl TM buffer containing 5 mgFast Red TR salt (Sigma).


"3 Incubate samples for 5–15 min at 37°C.Caution: Monitor the enzyme reactionunder a microscope and adjust thereaction time to keep the precipitatefrom becoming so dense that it shieldsnucleic acid sequences from the ISHdetection step.

"4 Wash samples 3 x 5 min with PBS.Note: Do not dehydrate samples afterwashing.

IIIB. !-Gal-BCIG reaction (for producing a blue precipitate visible under brightfieldmicroscopy)"1 Mix color reagent:

! 2.5 µl 5-bromo-4-chloro-3-indolyl-!-D-galactoside (BCIG; X-Gal) stocksolution [20 mg/ml BCIG (X-Gal) inN ,N -dimethylformamide].

! 100 µl diluent (PBScontaining 0.9 mMMgCl2, 3 mM potassium ferricyanide,and 3 mM potassium ferrocyanide).


"3 Incubate samples for 15–60 min at 37°C.Caution: Monitor the enzyme reactionunder a microscope and adjust thereaction time to keep the precipitatefrom becoming so dense that it shieldsnucleic acid sequences from the ISHdetection step.

"4 Wash samples 3 x 5 min with PBS.Note:I f you wish, dehydrate the samplesafter washing.

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Figure 2: Combined ICC and fluo-rescence ISH on EPLC 65 cells,showing the nuclear proliferationmarker Ki67, chromosome 7, andincorporated BrdU. Panel a: TheKi67 antigen (red) was visualizedwith a rabbit anti-Ki67 Ab, alkalinephosphatase-conjugated swine anti-rabbit Ab, and the APase-Fast Redreaction. The centromere ofchromosome 7 (green) wasvisualized directly with a FITC-labeled probe. Panel b: IncorporatedBrdU (blue) was visualized withmonoclonal anti-BrdU Ab,biotinylated horse anti-mouse Ab,and avidin-AMCA.

For brightfield microscopy (for detectionof !-Gal-BCIG ICC and enzyme-basedISH):

Depending on the enzyme reactions used,embed specimens in one of the following:! Aqueous, organic, or protein embedding

medium, as described in the article“Multiple target DN A in situ hybridi-zation with enzyme-based cytochemicaldetection systems” on page 100 of thismanual.

! Entellan (Merck) organic embeddingmedium and immersion oil (Zeiss) (forperoxidase-DAB or peroxidase-TMBreactions only).

! Tris-glycerol mixture [1:9 (v/v) mix of200 mM Tris-H Cl (pH 7.6) and glycerol](for peroxidase-DAB or phosphatase-Fast Red reactions only).

Results

VI. Fluorescence detection of BrdU(optional)"1 After performing the APase-Fast Red

ICC (Procedure IIIA) and fluorescenceISH (Procedure V) reactions on BrdU-labeled cells, incubate the cells for 45 minat room temperature with a specific anti-BrdU antibody.

"2 Wash slides for 2 x 5 min with PBScontaining 0.05% Tween® 20.

"3 Incubate samples with a secondaryantibody that is conjugated to a fluoro-chrome not used in the ICC or ISHreactions. Example: Use an alkaline-phosphataseconjugated antibody and the APase-Fast Red for ICC; a FITC-conjugatedantibody for ISH ; and an AMCA-conjugated antibody for BrdU detection.

Note: I f necessary, use an amplifiedthree-antibody detection procedure asoutlined in the article “Multiple targetDN A in situ hybridization with enzyme-based cytochemical detection systems”on page 100 of this manual.

"4 Before embedding, wash slides as follows:! 2 x 5 min with PBS containing 0.05%

Tween® 20.! 5 min with PBS.

"5 Include appropriate controls to ensurethat the BrdU detection step does notcross-react with ISH detection.

VII. EmbeddingFor fluorescence microscopy (for detec-tion of APase-Fast Red ICC, fluorescenceISH, and BrdU):

Embed specimens in a Tris-glycerol mix-ture [1:9 (v/v) mix of 0.2 M Tris-H Cl (pH7.6) and glycerol] containing 2% DABCO(Sigma) and (optionally) 0.5 µg/ml blueDAPI (Sigma).

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Figure 1: Combined ICC and fluorescence ISH on lung tumor cell line EPLC65, showingcytokeratin filaments, chromosome 1, andchromosome 17. The cytokeratins (red) werevisualized with a monoclonal anti-cytokeratinantibody (Ab), an alkaline phosphatase-conjugated goat anti-mouse Ab, and the APase-Fast Redreaction. The centromere of chromosome 1 (blue)was visualized with a biotinylated probe, avidin-AMCA, a biotinylated goat anti-avidin Ab, andavidin-AMCA. The centromere of chromosome 17(green) was visualized directly with a FITC-labeledprobe.

a b

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Strehl, S.; Ambros, P. F. (1993) Fluorescence in situhybridization combined with immunohistochemistryfor highly sensitive detection of chromosome 1aberrations in neuroblastoma. Cytogenet. CellGenet. 63, 24–28.Van Den Berg, H.; Vossen, J. M.; Van Den Bergh, R. L.; Bayer, J.; Van Tol, M. J. D. (1991) Detection of Y chromosome by in situ hybridization incombination with membrane antigens by two-colorimmunofluorescence. Lab. Invest. 64, 623–628.Van Den Brink, W.; Van Der Loos, C.; Volkers, H.;Lauwen, R.; Van Den Berg, F.; Houthoff, H.; Das, P. K.(1990) Combined !-galactosidase and immunogold/silver staining for immunohistochemistry and DNA in situhybridization. J. Histochem. Cytochem. 38, 325–329.Weber-Matthiesen, K.; Deerberg, J.; Müller-Hermelink, A.; Schlegelberger, B.; Grote, W.(1993) Rapid immunophenotypic characterization ofchromosomally aberrant cells by the new fictionmethod. Cytogenet. Cell. Genet. 63, 123–125.Zheng, Y.-L.; Carter, N. P.; Price, C. M.; Colman, S. M.;Milton, P. J.; Hackett, G. A.; Greaves, M. F.; Ferguson-Smith, M. A. (1993) Prenatal diagnosis from maternalblood: simultaneous immunophenotyping and FISH offetal nucleated erythrocytes isolated by negativemagnetic cell sorting. J. Med. Genet. 30, 1051–1056.

ReferencesHerbergs, J.; De Bruine, A. P.; Marx, P. T. J.;Vallinga, M. I. J.; Stockbrügger, R. W.; Ramaekers,F. C. S.; Arends, J. W.; Hopman, A. H. N. (1994)Chromosome aberrations in adenomas of the colon. Proof of trisomy 7 in tumor cells by combinedinterphase cytogenetics and immunocytochemistry. Int. J. Cancer 57, 781–785.Knuutila, S.; Nylund, S. J.; Wessman, M.;Larramendy, M. L. (1994) Analysis of genotype andphenotype on the same interphase or mitotic cell. A manual of MAC (morphology antibody chromo-somes) methodology. Cancer Genet. Cytogenet. 72,1–15.Mullink, H.; Walboomers, J. M. M.; Tadema, T. M.;Jansen, D. J.; Meijer, C. J. L. M. (1989) Combinedimmuno- and non-radioactive hybridocytochemistryon cells and tissue sections: influence of fixation,enzyme pre-treatment, and choice of chromogen on detection of antigen and DNA sequences. J. Histochem. Cytochem. 37, 603–609.Speel, E. J. M.; Herbergs, J.; Ramaekers, F. C. S.;Hopman, A. H. N. (1994a) Combined immunocyto-chemistry and fluorescence in situ hybridization forsimultaneous tricolor detection of cell cycle,genomic, and phenotypic parameters of tumor cells.J. Histochem. Cytochem. 42, 961–966.Speel, E. J. M.; Jansen, M. P. H. M.; Ramaekers, F. C. S.; Hopman, A. H. N. (1994b)Anovel triple-colordetection procedure for brightfield microscopy,combining in situ hybridization with immunocyto-chemistry. J. Histochem. Cytochem. 42, 1299–1307.Speel, E. J. M.; Ramaekers, F. C. S.; Hopman, A. H. N.(1995) Detection systems for in situ hybridization,and the combination with immunocytochemistry.Histochem. J., in press.

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Figure 3: Combined ICC and enzyme-based ISH on a human umbilical vein endothelial cell, showingthe intermediate filament protein vimentin, chromosome 1, and chromosome 7. Vimentin (blue) wasvisualized with monoclonal anti-vimentin Ab, !-galactosidase-conjugated goat anti-mouse Ab, and the !-galactosidase-BCIG reaction. The centromeres of chromosome 1 (biotinylated probe, brown) andchromosome 7 (digoxigenin-labeled probe, red) were visualized with avidin-peroxidase, rabbit anti-digoxigenin Ab and alkaline phosphatase-conjugated swine anti-rabbit Ab, the APase-Fast Red reaction, andthe PO-DAB reaction. Samples were embedded in a Tris-glycerol mixture, but were not counterstained.Slides were viewed by brightfield microscopy.

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Reagent Description Cat. No. Pack size 5-Bromo-2'-deoxy- One tablet contains approx. 50 mg 586 064 50 tabletsuridine 5-bromo-2'-deoxy-uridine and

approx. 20 mg binder.Pepsin Aspartic endopeptidase with broad 108 057 1 g

specificity 1693 387 5 gDigoxigenin-11-dUTP, Tetralithium salt, 1 mM solution 1093 088 25 nmolalkali-stable (25 µl)

1558 706 125 nmol(125 µl)

1570 013 5x125 nmol(5x125 µl)





104 493 1000 unitsAnti-Digoxigenin-AP Fab Fragments from sheep 1093 274 150 U

(200 µl)

II. In situ hybridization!1 Label a suitable probe DN A with dig-

oxigenin (DIG), using any of the proto-cols given elsewhere in this manual.

!2 Prepare hybridization solution whichcontains 60% deionized formamide, 300 mM N aCl, 30 mM sodium citrate,10 mM EDTA, 25 mM N aH 2PO 4 (pH7.4), 5% dextran sulfate, and 250 ng/µlsheared salmon sperm DN A.

!3 Denature the DIG-labeled probe DN Aat 80°C shortly before use and add it tothe hybridization solution at a concen-tration of 5 ng/µl.

!4 Add 10 µl of the hybridization mixture(hybridization solution plus denaturedprobe) to the fixed, permeabilized cellsand cover with an 18 x 18 mm coverslip.Note: An in situ denaturation step isoptional. Inclusion of such a step mayintensify the RN A signal since it makesthe sample more accessible to the probe.

!5 H ybridize at 37°C for 16 h.

III. Washes!1 After hybridization, remove coverslips

by shaking the slides at room tempera-ture in a solution of 60% formamide,300 mM N aCl, and 30 mM sodiumcitrate .

!2 Using the same formamide-salt solutionas in Step 1, wash the slides as follows:" Wash 3 x at room temperature." Wash 1x at 37°C.

!3 Finally, wash the slides 1 x 5 min in PBS.

The protocol given below has been devel-oped with a cell line (rat 9G) which hasintegrated into its genome the majorimmediate early transcription unit 1 ofhuman cytomegalovirus (Boom et al., 1986).The protocol is written in general terms andis applicable to abundantly expressedmRN A species. More information con-cerning this specific in situ hybridizationapplication can be found in Raap et al.(1991).

I. Cell preparation, fixation and permeabilizationNote: All solutions must be treated w ithRN ase inhibitors.!1 Culture cells at 37° C in a 5% CO 2

atmosphere on poly-L-lysine coatedmicroscopic slides. As culture medium,use Dulbecco’s minimal essential mediumwithout phenol red.

!2 Wash the cells with PBS at 37°C, thenfix at room temperature for 30 min in asolution of 4% (w/v) formaldehyde, 5%(v/v) acetic acid, and 0.9% (w/v) N aCl.

!3 Wash the fixed cells with PBS at roomtemperature and store them in 70%ethanol at 4°C.

!4 Before in situ hybridization, treat thefixed cells as follows:" Dehydrate by incubating successively

in 70% , 90% , and 100% ethanol." Wash in 100% xylene to remove

residual lipids." Rehydrate by incubating successively

in 100% , 90% , and 70% ethanol." Finally, incubate in PBS.

!5 Treat the fixed cells at 37°C with 0.1%(w/v) pepsin in 0.1 N H Cl, to increasepermeability to macromolecular reagents.

!6 Finally, treat the fixed cells as follows:" Wash with PBS for 5 min." Post-fix with 1% formaldehyde for

10 min." Wash again with PBS.

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In situhybridization to mRNA in in vitroculturedcells with DNA probesDepartment of Cytochemistry and Cytometry, University of Leiden, The N etherlands.

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!7 Embed the cell samples in an anti-fadingsolution which contains:" 9 parts glycerol

plus" 1 part staining mixture: 1 M Tris-

H Cl (pH 7.5), 2% 1,4-diaza-bicyclo-[2,2,2]-octane (DABCO ), and a DN Acounterstain [either propidium iodide(500 ng/ml) or DAPI (75 ng/µl)].

Results

IV. Immunofluorescent detectionNote: O ther protocols for amplifying thesignal may also be used. See the protocolsunder “Single color fluorescent detectionwith immunological amplification” onpage 62.!1 Block non-specific binding by the

following steps:" Add 100 µl blocking solution

[100 mM Tris-H Cl; pH 7.5, 150 mMN aCl, 0.5% (w/v) blocking reagent]to each slide.

" Cover with a 24 x 50 mm coverslip." Place slide in a moist chamber.

!2 To loosen the coverslips, wash the slidesbriefly with the buffer used for immuno-logical detection.

!3 Detect the hybridized DIG-labeledprobe by incubating the slides in a moistchamber for 45 min with a 1:500 dilu-tion of anti-DIG-fluorescein in blockingsolution (from Step 1) .

!4 Wash slides with a solution of 100 mMTris-H Cl, pH 7.5; 150 mM N aCl;0.05% Tween® 20.

!5 To dehydrate the cell samples, incubatethe slides for 5 min in each of a series of ethanol solutions: 70% , 90% , then100% ethanol.

!6 Air dry the slides.

Panel a: PI counterstain.

Panel b: DAPI counterstain.

Figure 1: Nuclear staining of integrated IE viral DNA after induction with cycloheximide. The signal throughout the cytoplasm represents viralmRNA. The two panels show the same signal withdifferent counterstains.

ReferencesBoom, R.; Geelen, J. L.; Sol, C. J.; Raap, A. K.;Minnaar, R. P.; Klaver, B. P.; Noordaa, J. v. d. (1986)Establishment of a rat cell line inducible for theexpression of human cytomegalovirus immediate –early gene products by protein synthesis inhibition.J. Virol. 58, 851–859.Raap, A. K.; Van de Rijke, F .M.; Dirks, R. W.; Sol, C. J.;Boom, R.; Van der Ploeg, M. (1991) Bicolor fluores-cence in situ hybridization to intron- and exonmRNA sequences. Exp. Cell Res. 197, 319–322.

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Reagent Description Cat. No. Pack size Pepsin Aspartic endopeptidase with broad 108 057 1 g

specificity 1693 387 5 gTween® 20 1332 465 5x10 mlBlocking Reagent Powder 1096 176 50 gAnti-Digoxigenin Fab Fragments from sheep 1207 741 200 µgFluoresceinDAPI Fluorescence dye for staining of 236 276 10 mg

chromosomesDigoxigenin-11-dUTP, Tetralithium salt, 1 mM solution 1093 088 25 nmolalkali-stable (25 µl)

1558 706 125 nmol(125 µl)

1570 013 5x125 nmol(5x125 µl)





104 493 1000 unitsEDTA 808 261 250 g

808 270 500 g808 288 1 kg

II. Cell fixation and preparation of cell smearsNote: This procedure was adapted fromAmann et al., 1990.!1 Fix the cells by adding 3 volumes of para-

formaldehyde solution (4% paraform-aldehyde in PBS) to 1volume of suspendedcells.

!2 After 3 h incubation, do the following:" Pellet cells by centrifugation." Remove the supernatant." Wash the cell pellet with PBS." Resuspending the cells in an aliquot

of PBS.Note: After adding 1 volume ethanol tothe resuspended cells, you may store thecell suspension at –20°C for up to 3months w ithout apparent influence onthe hybridization results.

!3 Spot these fixed cell suspensions ontothoroughly cleaned glass slides andallow to air dry for at least 2 h.

!4 Dehydrate the cell samples by immers-ing the slides successively in solutions of50% ethanol, then 80% ethanol, then98% ethanol (3 min for each solution).

III. DIG labeling of oligonucleotideswith DIG-ddUTPLabel oligonucleotides either according tothe protocols in Chapter 4 of this manualor at the 5' end according to the protocolof the DIG O ligonucleotide 5' -End Label-ing Set*.

Note: An earlier version of this procedurehas been published (Zarda et al., 1991).

I. Organisms and growth conditions!1 To prepare cells needed for this experi-

ment, do the following:" Allow Escherichia coli (Deutsche

Sammlung von Mikroorganismen undZellkulturen GmbH , DSM 30083) togrow aerobically in YT broth (tryptone,10 g/L; yeast extract, 5 g/L; glucose,5 g/L; sodium chloride, 8 g/L; pH 7.2)at 37°C.

" Cultivate Pseudomonas cepacia (DSM50181) aerobically in M1 broth(peptone, 5 g/L; malt extract, 3 g/L;pH 7.0) at 30°C.

Note: Cells from Methanococcus vannielii(DSM1224) were a generous gift of Dr. R . H uber (Dept. of Microbiology,University of Regensburg, FRG).

!2 To guarantee a high cellular rRN Acontent, harvest all cells at mid-logarithmic phase by centrifugation in amicrocentrifuge (5000 x g, 1 min).

!3 Discard the growth medium from thecell pellet and resuspend cells thoroughlyin phosphate buffered saline (130 mMsodium chloride, 10 mM sodium phos-phate; pH 7.2).

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Identification of single bacterial cells using DIG-labeled oligonucleotidesB. Zarda, Dr. R . Amann, and Prof. Dr. K.-H . Schleifer, Institute for Microbiology, Technical University of Munich, Germany.

*Sold under the tradename of Genius in the US.

!5 View the cell smears with a Plan-N eofluar 100 x objective (oil immersion)on the following setup: a Zeiss Axioplanmicroscope fitted for epifluorescencemicroscopy with a 50 W mercury highpressure bulb and Zeiss filter sets 09 and15 (Zeiss, O berkochen, FRG).

!6 For photomicrographs, use Kodak Ekta-chrome P1600 color reversal film and an exposure time of 15 s for epifluores-cence or 0.01 s for phase contrastmicrographs.

Vb. Detection of DIG-labeledoligonucleotides with peroxidase-conjugated anti-DIG Fab fragments!1 Use the same conditions for hybridiza-

tion and antibody binding as for fluores-cent antibodies (Procedures IV and Va),except include a lysozyme pretreatmentof the cell smears prior to hybridization.For details, see Procedure IV above.

!2 Visualize the bound antibody as follows:" Prepare peroxidase substrate-nickle

chloride solution [1.3 mM diamino-benzidine, 0.02% (v/v) H 2O 2, 0.03%(w/v) nickel chloride, 5 mM Tris-H Cl(pH 7.4)].

" Incubate slide with peroxidase sub-strate-nickle chloride solution until apurplish-blue precipitate forms.

!3 View slides under a light microscope andphotograph with Kodak EktachromeP1600 color reversal film.

ResultsUsing the techniques in this article, wecould detect P. cepacia cells in a mixedsample of three bacteria (Figure 1). Addi-tional experiments with a DIG-labeledoligonucleotide not complementary torRN A show no significant levels of non-specifically bound DIG-labeled nucleic acidprobes and anti-DIG antibodies.

IV. In situ hybridization using digoxigenin-labeled oligonucleotides!1 Prepare hybridization solution (900 mM

sodium chloride, 20 mM Tris-H Cl,0.01% sodium dodecyl sulfate; pH 7.2).

!2 Depending on the method of analysis,do either of the following:" I f you will use fluorescently-labeled

antibodies (Procedure Va below),proceed directly to Step 3.

" I f you will use peroxidase-conjugatedantibodies (Procedure Vb below), then:

" Prior to hybridization, incubate fixedcells for 10 min at 0°C with 1 mg/mlof lysozyme in TE (100 mM Tris-H Cl, 50 mM EDTA; pH 8.0).

" Remove lysozyme by thoroughlyrinsing the slide with sterile H 2O .

" Air dry the slide." Proceed to Step 3.

!3 Add 8 µl hybridization solution contain-ing 50 ng of labeled oligonucleotideprobe to the prepared slide (fromProcedure II).

!4 Incubate the slide for 2 h at 45°C in anisotonically equilibrated humid chamber.

Va. Detection of DIG-labeled oligonucleotides with fluorescently labeled anti-DIG Fab fragments!1 Dilute fluorescein- or rhodamine-labeled

anti-DIG Fab fragments 1:4 in blockingsolution (150 mM sodium chloride;100 mM Tris-H Cl, pH 7.5; 0.5% bovineserum albumin; and 0.5% blockingreagent).

!2 Add 10 µl of the diluted antibody to theslide and incubate the slide for anotherhour at 27°C in the humid chamber.

!3 Remove the slide from the humid cham-ber and immerse in 40 ml of a washingsolution (150 mM sodium chloride,100 mM Tris-H Cl, 0.01% SDS; pH 7.4)at 29°C for 10 min.

!4 Prepare the slides for viewing by doingthe following:" Rinse the slides briefly with sterile

water (which has been filtered througha 0.2 µm filter).

" Air dry." Mount in Citifluor (Citifluor Ltd.,

London, U.K.).

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5

Figure 1: Specific identification of whole fixed cells of P. cepaciain a mixture of P. cepacia (chainsof rods), E. coli (rods) and M. van-nielii (cocci) with a DIG-labeled oligonucleotide probe.Phase contrast (panel a) andepifluorescence (panel b) photosshow detection with fluorescentlylabeled anti-DIG Fab fragments(Zarda et al., 1991). Phase contrast(panel c) and brightfield (panel d)photos show detection withperoxidase-conjugated anti-DIG Fabfragments

Reprinted from Zarda, B.; Amann, R.;Wallner, G.; Schleifer, K. H. (1991) Iden-tification of single bacterial cells using digoxigenin-labeled rRNA-targetedoligonucleotides. J. Gen. Microbiol. 137,2823–2830 with permission of thesociety for general microbiology.

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ReferencesAmann, R. I.; Binder, B. J.; Olson, R. J.; Chisholm,S. W.; Devereux, R.; Stahl, D. A. (1990) Combinationof 16 S rRNA-targeted oligonucleotide probes withflow cytometry for analyzing mixed microbial popu-lations. Appl. Environ. Microbiol. 56, 1919–1925.Mühlegger, K.; Huber, E.; von der Eltz, H.; Rüger, R.;Kessler, C. (1990) Nonradioactive labeling anddetectionof nucleic acids. Biol. Chem. Hoppe-Seyler371, 953–965.

a b

c d

Zarda, B.; Amann, R.; Wallner, G.; Schleifer, K. H.(1991) Identification of single bacterial cells usingdigoxigenin-labeled rRNA-targetedoligonucleotides.J. Gen. Microbiol. 137, 2823–2830.


Reagent Description Cat. No. Pack size DIG-Oligonucleotide 3'-end labeling of oligonucleotides from 1362 372 1 Kit3'-End Labeling Kit* 14 to 100 nucleotides in length with (25 labeling

DIG-11-ddUTP and terminal transferase reactions)DIG-Oligonucleotide With theDIGOligonucleotide5'-EndLabeling 1480 863 1 Set5'-End Labeling Set* Set, oligonucleotides can be labeled with (10 labeling

digoxigenin at the 5’-end after synthesis that reactions)includes the addition of a phosphoramidite

Anti-Digoxigenin- Fab Fragments from sheep 1207 741 200 µgFluoresceinAnti-Digoxigenin- Fab Fragments from sheep 1207 750 200 µgRhodamineAnti-Digoxigenin-POD Fab Fragments from sheep 1207 733 150 UTris Powder 127 434 100 g

708 968 500 g708 976 1 kg

EDTA Powder 808 261 250 g808 270 500 g808 288 1 kg

Blocking Reagent Powder 1096 176 50 gBSA Highest quality lyophilizate 238 031 1 g

238 040 10 gSDS Purify 99% 1028 685 100 g

1028 693 500 g *Sold under the tradename of Genius in the US.

#

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Procedures for In Situ H ybridization to Chromosomes, Cells, and Tissue SectionsISH to Tissues

5

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! The method is economical; the DIGLabeling and Detection Kit* is sufficientfor preparing 10 ml of DIG-labeled probecocktail (1 µl labeled DN A/ml). This isenough for hybridizing 1000 sections.

! Problems linked to endogenous biotin areavoided, since endogenous digoxigeninapparently does not exist.

The main steps in performing in situ hy-bridization on formalin-fixed, paraffin-embedded specimens are:"1 Exposure of target DN A. This is done

with a carefully controlled proteolyticdigestion step.

"2 Denaturation of ds DN A, followed byhybridization.

"3 Washing and detection of DN A hybrids.

In our experience the most difficult step isoptimization of the proteolytic digestion.

I. Probe preparation"1 Using standard methods, remove the viral

insert from its vector by restrictionenzyme cleavage and separate it on asubmerged agarose gel.

"2 Recover the insert from the gel byelectroelution or other methods.

"3 Label the 8 kb viral insert nonradioac-tively with digoxigenin acccording to theprocedures given in Chapter 4 of thismanual.

"4 To a tube, add the following ingredientsof the probe cocktail:! 10 µl 50 x Denhart’s solution.! 50 µl dextran sulfate 50% (w/v).! 10 µl sonicated salmon sperm DN A

(10 mg/µl).! 100 µl 20 x SSC.! 500 ng digoxigenin-labeled probe in

50 µl TE.! Enough distilled water to make a total

volume of 250 µl."5 To complete the probe cocktail, add

250 µl formamide to the tube.Tip: Use gloves when handling solu-tions containing formamide. Formamideshould be handled in a fume hood.

"6 Mix the probe cocktail (by vortexing) andstore it at –20°C.

The technique of tissue in situ hybridizationhas become a powerful tool in pathology,although it is still mainly used for researchpurposes. The most important application atthe moment is probably the demonstrationof viral DN A (or RN A) in biopsies. Thistechnique has made it possible todemonstrate infection with CMV (Grody etal., 1987; Unger et al., 1988), H BV (Blum etal., 1983; N egro et al., 1985), parvovirus(Proter et al., 1988), coxsackie B (Archard etal., 1987) EBV and H IV (Pezzella et al.,1989). In situ hybridization has been widelyused to demonstrate H PV sequences inbiopsies from the uterine cervix as well asfrom the head and neck (Lindeberg et al.,1990; Lindeberg et al., 1989; Shah et al.,1988). Although some of these viruses can bedemonstrated by other methods, in situhybridization is much faster than methodsusing infection of tissue cultures; for H PVtissue culture methods do not exist.

Radioactively labeled probes were usedoriginally, but in recent years nonradio-active probes have become increasinglypopular for obvious reasons.

In general, the use of radioactively labeledprobes is considered superior to othermethods. H owever, this is probably notcorrect (N uevo and Richart, 1989). Forinstance, H PV type 16 was demonstrated inSiH a cells by in situ hybridization withdigoxigenin-labeled probes (H eiles et al.,1988). As SiH a-cells contain only 1–2 H PVcopies per cell, one can hardly ask for afurther improvement in sensitivity.

There is an increasing demand from histo-pathologists for simple in situ hybridizationmethods, so that laboratory technicians canperform, for example, typing of H PV as adaily routine. To answer this demand, wepresent here a protocol for the detection ofH PV DN A type 11 in laryngeal papillomasas well as in genital condylomatous lesions.The method is used on routine materialwhich has been fixed in formalin andembedded in paraffin. The method describedhas the following advantages:! The method is fast; results are obtained in

about 5–6 h, and the hands-on time isabout 2 h.

Detection of HPV 11 DNA in paraffin-embeddedlaryngeal tissue with a DIG-labeled DNA probeDr. J. Rolighed and Dr. H . L indeberg, EN T-department and Institute for Pathology, Aarhus University H ospital, Denmark .


***Sold under the tradename ofGenius in the US.

***EP Patent 0 324 474 granted toBoehringer Mannheim GmbH.



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Nuevo, G. J.; Richart, R. M. (1989) A comparison ofBiotin and 35S-based in situ hybridization meth-odologies for detection of human papillomavirusDNA. Lab. Invest. 61, 471–475.Pezzella, M.; Pezzella, F.; Rapicetta, M. et al. (1989)HBV and HIV expression in lymph nodes of HIVpositive LAS patients: histology and in situ hybridi-zation. Mol. Cell Probes 3, 125–132.Proter, H. J.; Quantrill, A. M.; Fleming, K. A. (1988)B19 parvovirus infection of myocardial cells. Lancet 1, 535–536.Shah, K. V.; Gupta, J. W.; Stoler, M. H. (1988) The in situ hybridization test in the diagnosis ofhuman papillomaviruses. In: De Palo, G.; Rilke, F.;zur Hausen, H. (Eds.) Serono Symposia 46: Herpesand Papillomaviruses. New York: Raven Press,151–159.Unger, E. R.; Budgeon, L. R.; Myorson, B.; Brigati, D.J. (1988) Viral diagnosis by in situ hybridization.Description of a rapid simplified colorimetricmethod. Am J. Surg. Pathol. 18, 1–8.

ReferencesArchard, L. C.; Bowles, N. E.; Olsen, E. G. J.;Richardson, P. J. (1987) Detection of persistentcoxsachie B virus RNA in dilated cardiomyopathyand myocarditis. Eur. Heart J. (Suppl.) 8, 437–440.Blum, H. E.; Stowringer, L.; Figue, A. et al. (1983)Detection of Hepatitis B virus DNA in hepatocytes,bile duct epithelium, and vascular elements by in situ hybridization. Proc. Natl. Acad. Sci. (USA)80, 6685–6688.Grody, W. W.; Cheng, L.; Lewin, K. J. (1987) In situviral DNA hybridization in diagnostic surgicalpathology. Hum. Pathol. 18, 535–543.Heiles, H. B. J.; Genersch, E.; Kessler, C.; Neumann,R.; Eggers, H. J. (1988) In situ hybridization withdigoxigenin-labeledDNA of human papillomaviruses(HPV 16/18) in HeLa and SiHa cells. BioTechniques6 (10), 978–981.Lindeberg, H.; Johansen, L. (1990) The presence of human papillomavirus (HPV) in solitary adultlaryngeal papillomas demonstrated by in situDNA hybridization with sulphonated probes. Clin.Otolaryngol. 15, 367–371.Lindeberg, J.; Syrjänen, S.; Karja, J.; Syrjänen, K.(1989) Human papillomavirus type 11 DNA insquamous cell carcinomas and preexisting multiplelaryngeal papillomas. Acta Oto-Laryngol. 107,141–149.Negro, F.; Berninger, M.; Chaiberge, E. et al. (1985)Detection of HBV-DNA by in situ hybridization usinga biotin-labeled probe. J. Med. Virol. 15, 373–382.

Reagent Description Cat. No. Pack size DIG DNA Labeling and Random primed labeling of DNA probes 1093 657 1 Kit (25 labelingDetection Kit*, **, *** with DIG-11-dUTP, alkali-labile and color reactions and

detection of DIG-labeled hybrids. detection of 50blots of 100 cm2)

Proteinase K Lyophilizate 161 519 25 mg745 723 100 mg

1000 144 500 mg1092 766 1 g

Tris Powder 127 434 100 g708 968 500 g708 976 1 kg

EDTA Powder 808 261 250 g808 270 500 g808 288 1 kg

Blocking Reagent Powder 1096 176 50 gAnti-DIG-AP Fab Fragments from sheep 1093 274 150 U (200 µl)NBT/BCIP Stock solution 1681 451 8 ml

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IV. Hybridization"1 After the treatment with Proteinase K,

remove the coverslips and fix the sectionsin 0.4% formaldehyde for 5 min at 4°C.

"2 Immerse the sections in distilled water for5 min.

"3 Allow the sections to drip and air dry for5 min.

"4 Distribute about 5–10 µl of the probecocktail over each section. Note: Large biopsies may require largervolumes of probe cock tail.

"5 Prepare a negative control by coveringone section from each block with a“blind” probe cocktail, i.e. a probecocktail containing all the ingredients inProcedure I, except the labeled H PVDN A.

"6 Place coverslips over each section anddenature the DN A by placing the slideson a heating plate at 95°C for 6 min.Tip: We use only siliconized coverslips.

"7 Cool the slides for 1 min on ice."8 Place the slides in a humid chamber and

incubate for 3 h at 42°C in an oven.Tip: The hybridization time may beprolonged overnight when convenient.

V. WashesRemove the coverslips and wash the sectionsas follows:

! 2 x 5 min in 2 x SSC at 20°C.! 1 x 10 min in 0.1 x SSC at 42°C.

II. Pretreatment of slides"1 Wash slides overnight in a detergent, fol-

lowed by an intensive wash in tap water,and finally, a wash in demineralized water.

"2 After drying the slides, wash them for 3 min in acetone.

"3 Transfer the washed slides to a 2%solution of 3-aminopropyltrithoxysilanein acetone and incubate for 5 min.

"4 Wash the slides briefly in distilled waterand allow to dry.Note:The coated slides can be stored dryunder dust-free conditions for severalmonths.

III. Preparation of tissue sections"1 Fix biopsies from condylomatous lesions

and from laryngeal papillomas in 4%phosphate-buffered formaldehyde andembed them in paraffin.

"2 Cut routine sections, float them ondemineralized water and place them onthe pretreated slides.

"3 Treat the sections on the slides as follows:! Bake the sections for 30 min at 60°C.! Dewax in xylene.! Rehydrate them by dipping them in

a graded ethanol series (2 x in 99% ethanol, 2 x in 96% ethanol, 1x in 70%ethanol, 1x in H 2O ).

"4 Immediately before use, prepare a workingProteinase K solution as follows:! Thaw an aliquoted frozen stock

solution of Proteinase K, 10 mg/ml, inH 2O .

! Dilute the stock Proteinase K to theworking concentration of 100 µg/ml inTES [50 mM Tris-H Cl (pH 7.4), 10 mM EDTA, 10 mM N aCl].

"5 Treat each section with 20–30 µl of theworking solution of Proteinase K for 15 min at 37°C. During the proteolyticdigestion cover the sections with cover-slips and place them in a humid chamber. Tip: We use only siliconized coverslips.

Note: The proteolytic treatment iscrucial and may need to be varied w ithdifferent tissues.When too muchProteinase K is used, the tissue totallydisintegrates, and if too little is usedpositive signals may be totally absent.

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5

"10 After the incubation, do the following:! Remove the coverslips.! Wash the sections gently.! Stain a few seconds with neutral red.! Mount.

Caution: Avoid dehydrating proce-dures since ethanol may remove orweaken the signal.

ResultsThe described method is applied to biopsiesof multiple and solitary laryngeal papillomasand on a flat condylomatous lesion of theuterine cervix (Figure 1). The positive resultsare obvious, and little background isobserved in these examples. The procedureis extremely straightforward and can beperformed in 5–6 h, including the 3 hhybridization. Consequently, the wholeprocedure can be performed in 1 day andthe results recorded the next morning. Ifneeded, the hybridization can be shortened;in our initial experiments, we were able todetect H PV type 11 after only 1 h ofhybridization.

AcknowledgmentPlasmids with H PV type 11 were kindlysupplied by Prof. zur H ausen and co-workers,DKFZ, H eidelberg, Germany.

VI. Detection of hybrids"1 Dip each section into buffer 1 [100 mM

Tris-H Cl,150mM N aCl;pH 7.5 (20°C)]."2 Cover each section with 20–40 µl buffer 2

[0.5% (w/v) blocking reagent in buffer 1],then add a coverslip.

"3 Incubate sections at 20°C for 15 min."4 Remove the coverslips and dip the

sections in buffer 1."5 Dilute the antibody conjugate 1:500 in

buffer 2."6 Distribute 10–20 µl of the diluted con-

jugate over each section, including thenegative controls.

"7 Place a coverslip over each section andplace all sections in a humid chamber atroom temperature for 1 h.

"8 Remove the coverslips, wash the sections2 x10 min in buffer 1, then equilibrate for5 min in buffer 3.

"9 Distribute approx. 20 µl color-solution(N BT/BCIP) onto each section, coverwith a coverslip and leave in the darkovernight. Note: You may shorten the color-reaction to 30–60 min if you w ish. Infact, a positive reaction may be seenafter a few minutes in the dark whenH PV is present in a high copy-number.

Hint: Use gloves when handling thecolor solution.

Figure 1: Detection of HPV 11 DNA in biopsies of multiple and solitary laryngeal papillomas and on a flat condylomatous lesion of the uterine cervix. Positive results are shown in Panels a and c. The negative controls (Panels b and d) show no signal.

a b

c d#

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The protocols below are written in generalterms. Additional information concerningthe in situ hybridization methods andimportant technical details are provided inKomminoth (1992), Komminoth et al. (1992),Komminoth et al. (1995), Sambrook et al.(1989), and the Boehringer MannheimDIG/Genius™ System User’s Guide forMembrane H ybridization.

I. Preparation of slidesNote:Gelatin-coated slidesare excellent forlarge tissue sections obtained from frozenor paraffin-embedded specimens. A lterna-tively, silane-coated slides can be used, butare better suited for small tissue samples orcell preparations.

IA. Gelatin-coated slides!1 Clean slides by soaking for 10 min in

Chromerge® solution (Merck).!2 Wash slides in running hot water.!3 Rinse slides in distilled water.!4 Prepare gelatin solution as follows:

" Dissolve 10 g gelatin (Merck) in 1 literof distilled water which has beenheated to 40°–50°C.

" Add 4 ml 25% chromium potassiumsulfate (CPS) solution (Merck) to thegelatin to make a final concentration of0.1% CPS.

!5 Dip slides in gelatin solution for 10 min.!6 Let the slides air dry.!7 Soak slides for 10 min in PBS (pH 7.4)

containing 1% paraformaldehyde.!8 Let the slides air dry.!9 Bake slides at 60°C overnight.

IB. Silane-coated slides!1 Soak clean glass slides for 60 min in silane

solution [5 ml of 3-aminopropyltrieth-oxysilane (Sigma) in 250 ml of acetone].

!2 Wash the slides 2 x 10 min in distilledwater.

!3 Dry slides overnight at 60°C.

Note:Both gelatin- and silane-coated slidescan be stored for several months under dryand dust free conditions.

The protocols given below are modificationsof recently published methods for non-isotopic in situ hybridization with dig-oxigenin-labeled probes (Komminoth, 1992;Komminoth et al., 1992). In those reports wedemonstrated:

" Digoxigenin-labeled probes are as sensi-tive as 35S-labeled probes.

" Protocols with digoxigenin-labeled probesmay also be applied to diagnostic in situhybridization procedures.

The following protocols have been success-fully used in our laboratories to detectmRN A in frozen and paraffin-embeddedtissue sections with either RN A or oligo-nucleotide probes.

RN A probes are best for high sensitivitydetection procedures because:

" RN A probes are easily generated andlabeled by in vitro transcription procedures.

" H ybrids between mRN A and RN Aprobes are highly stable.

" Protocols using stringent washingconditions and RN ase digestion stepsyield highly specific signals with lowbackground.

Synthetic oligonucleotide probes are anattractive alternative to RN A probes for thedetection of abundant mRN A sequences intissue sections, because:

" Any known nucleic acid sequence canrapidly be made by automated chemicalsynthesis.

" Such probes are more stable than RN Aprobes.

O ligonucleotide probes are labeled duringsynthesis or by addition of reportermolecules at the 5' or 3' end after synthesis.The most efficient labeling method isaddition of a “tail” of labeled nucleotides tothe 3' end. O ligonucleotide probes aregenerally less sensitive than RN A probessince fewer labeled nucleotides can beincorporated per molecule of probe.

Detection of mRNA in tissue sections using DIG-labeled RNA and oligonucleotide probesP. Komminoth, D iv ision of Cell and Molecular Pathology, Department of Pathology,University of Zürich, Switzerland.

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IIB. Paraffin sections!1 Prepare formaldehyde-fixed and paraffin-

embedded material according to standardprocedures.

!2 Cut 7 µm sections from the paraffin-embedded material and place the sectionsonto coated glass slides (from Procedure I).

!3 Dry slides in an oven at 40°C overnight. !4 Dewax sections 2 x 10 min with fresh

xylene.!5 Rehydrate sections in the following

solutions:" 1 x 5 min in 100% ethanol." 1 x 5 min in 95% ethanol." 1 x 5 min in 70% ethanol." 2 x in DEPC-treated ddH 2O .

IIIA. ISH protocol for detection of mRNAwith DIG-labeled RNA probesCaution: Prepare all solutions for proce-dures below (probe labeling throughposthybridization) with distilled, deion-ized water (ddH 2O ) that has been treatedwith 0.1% diethylpyrocarbonate (DEPC)(Sambrook et al., 1989). To avoid RN asecontamination, wear gloves throughoutthe procedures and use different glasswarefor pre- and posthybridization steps.

Note: Perform all procedures below atroom temperature unless a differenttemperature is stated.

Probe labeling

Note: To ensure tissue penetration, we prefer to work w ith RN A probes that are≤ 500 bases long.!1 Clone a cDN A insert into an RN A

expression vector (plasmid) according tostandard procedures (Sambrook et al.,1989).

!2 Linearize the RN A expression vectorwith appropriate restriction enzymes toallow in vitro run-off synthesis of bothsense- and antisense-oriented RN A probes(Valentino et al., 1987).

Note: As an alternative to linearizedplasmids, use PCR-generated templatescontaining RN A polymerase promotersequences for in v itro transcription(Young et al., 1991).

II. Tissue preparationGeneral guidelines: Fix or freeze tissue assoon as possible after surgical excision toprevent degradation of mRN A.

If possible, use cryostat sections of para-formaldehyde-fixed tissues which have beenimmersed in sucrose (as in Procedure IIA).These provide excellent conditions formRN A localization and preservation ofsample morphology.

H owever, in surgical pathology, most tissuesare routinely fixed in formalin. For mRN Alocalization, do not fix in formalin for morethan 24 h. Prolonged storage of samples informalin will cause covalent linkagesbetween mRN A and proteins, making targetsequences less accessible.

Caution: Prepare all solutions for proce-dures I IA and I IB with distilled, deionizedwater (ddH 2O ) that has been treated with 0.1% diethylpyrocarbonate (DEPC)(Sambrook et al., 1989).

IIA. Frozen sections!1 Cut tissues into 2 mm thick slices.!2 Incubate cut tissues at 4°C for 2–4 h with

freshly made, filtered fixative (DEPC-treated PBS containing 4% paraformal-dehyde; pH 7.5).

!3 Decant fixative and soak tissues at 4°Covernight in sucrose solution [DEPC-treated PBS containing 30% sucrose(RN ase-free)].Note:During this step, the tissues shouldsink to the bottom of the container.

!4 Store tissue samples in a freezing com-pound at –80°C, or, for long time storage,at –140°C (N aber et al., 1992).

!5 For sectioning, warm the samples to –20°C and cut 10 µm sections in a cryostat.

!6 Place the sections on pretreated glassslides (from Procedure I).

!7 Dry slides in an oven at 40°C overnight.!8 Do either of the following:

" Use the prepared slides immediately.or" Store the slides in a box at –80°C.

Before processing, warm the storedslides to room temperature and drythem in an oven at 40°C for aminimum of 2 h.Note:Slides with cryostat sectionsmaybe stored at –80°C for several weeks.

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Caution: Permeabilization is the mostcritical step of the entire in situ hybridi-zation procedure. In paraffin-embeddedarchival materials, optimal tissue perme-abilization differs for each case,depending upon duration and type offixation. We recommend titration of theProteinase K concentration. A lternativepermeabilization protocols for improvedefficiency of digestion include incubationof slides for 20–30 min at 37°C with0.1% pepsin in 0.2 M H Cl.

!5 Post-fix sections for 5 min at 4°C withDEPC-treated PBS containing 4% para-formaldehyde.

!6 Wash sections 2 x 5 min with DEPC-treated PBS.

!7 To acetylate sections, place slide contain-ers on a rocking platform and incubateslides 2 x 5 min with 0.1 M triethanol-amine (TEA) buffer, pH 8.0, containing0.25% (v/v) acetic anhydride (Sigma).Caution: Acetic anhydride is highlyunstable. Add acetic anhydride to eachchange of TEA-acetic anhydride solu-tion immediately before incubation.

!8 Incubate sections at 37°C for at least 10 min with prehybridization buffer [4 x SSC containing 50% (v/v) deionizedformamide].Note: Deionize formamide w ithDowex® MR 3 (Sigma) according toprotocols described in Sambrook et al.(1989).

Note: 1 x SSC = 150 mM N aCl, 15 mMsodium citrate, pH 7.2.

In situ hybridization!1 Prepare hybridization buffer containing:

" 40% deionized formamide." 10% dextran sulfate." 1 x Denhardt’s solution [0.02% Ficoll®,

0.02% polyvinylpyrrolidone, 10 mg/mlRN ase-free bovine serum albumin].

" 4 x SSC." 10 mM DTT." 1 mg/ml yeast t-RN A." 1 mg/ml denatured and sheared

salmon sperm DN A.Caution: Denature and add salmonsperm DN A to buffer shortly beforehybridization.

Note: H ybridization buffer (minussalmon sperm DN A) can be stored at–20°C for several months.

!3 Purify linearized plasmid by phenol-chloroform extraction and ethanolprecipitation.Caution: Some plasmid purification k itscontain RN ase digestion steps for remov-ing bacterial RN A. Traces of RN ase maydestroy transcribed probes. To ensureremoval of residual RN ase, performmultiple phenol-chloroform extractions ofthe linearized plasmid.

!4 To avoid RN A polymerase inhibition,resuspend the plasmid in EDTA-freebuffer or DEPC-treated ddH 2O .

!5 Generate digoxigenin-labeled RN Aprobes in both the sense and antisensedirection by in vitro transcription withthe DIG RN A Labeling Kit* or accord-ing to procedures in Chapter 4 of thismanual.

!6 Purify labeled probes according toprocedures in the DIG/Genius™ SystemUser’s Guide for Membrane H ybridi-zation or in Chapter 4 of this manual.Caution: Probes longer than 500 basesmay not penetrate tissue. I f probes arelonger than 500 bases, shorten them by alkaline hydrolysis according to procedures in the DIG/Genius™ SystemUser’s Guide for Membrane H ybridi-zation.

!7 Estimate the yield of labeled probes bydirect blotting procedures as described inChapter 4 of this manual.

!8 Aliquot labeled probes and store them at–80°C.Note: Probes are stable for up to oneyear.

Prehybridization!1 Incubate sections as follows:

" 2 x 5 min with DEPC-treated PBS (pH7.4) (Sambrook et al., 1989).Note: PBS contains 140 mM N aCl,2.7 mM KCl, 10 mM N a2H PO 4,1.8 mM KH 2PO 4.

" 2 x 5 min with DEPC-treated PBScontaining 100 mM glycine.

!2 Treat sections for 15 min with DEPC-treated PBS containing 0.3% Triton®

X-100.!3 Wash 2 x 5 min with DEPC-treated PBS.!4 Permeabilize sections for 30 min at 37°C

with TE buffer (100 mM Tris-H Cl,50 mM EDTA, pH 8.0) containing either1 µg/ml RN ase-free Proteinase K (forfrozen sections) or 5–20 µg/ml RN ase-free Proteinase K (for paraffin sections).


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!1 Using a shaking platform, wash sections2 x 10 min with buffer 1 [100 mM Tris-H Cl (pH 7.5), 150 mM N aCl].

!2 Cover sections for 30 min with blockingsolution [buffer 1 containing 0.1%Triton® X-100 and 2% normal sheepserum (Sigma)].

!3 Decant blocking solution and incubatesections for 2 h in a humid chamber withbuffer 1 containing 0.1% Triton® X-100,1% normal sheep serum, and a suitabledilution of sheep anti-DIG-alkalinephosphatase [Fab fragments].Note: For optimal detection, incubateseveral sections (from the same sample)with different dilutions of the antibody(1:100; 1:500, and 1:1000).

!4 Using a shaking platform, wash sections2 x10 min with buffer 1.

!5 Incubate sections for 10 min with buffer 2[100 mM Tris-H Cl (pH 9.5), 100 mMN aCl, 50 mM MgCl2].

!6 Prepare a color solution containing:" 10 ml of buffer 2." 45 µl nitroblue tetrazolium (N BT)

solution (75 mg N BT/ml in 70%dimethylformamide).

" 35 µl 5-bromo-4-chloro-3-indolyl-phosphate (BCIP or X-phosphate)solution (50 mg X-phosphate/ml in100% dimethylformamide).

" 1 mM (2.4 mg/10 ml) levamisole (Sig-ma).Note: For convenience, prepare a 1 Mstock solution of levamisole in ddH 2O(stable for several weeks, when storedat 4°C) and add 10 µl of the stock to10 ml of the color solution. I f endog-enous phosphatase activ ity is high,increase the concentration oflevamisole to 5 mM (50 µl 1 Mstock /10 ml solution) in the colorsolution.

Note: N BT/BCIP produces a blue pre-cipitating product. For other colors, useother alkaline phosphatase substrates.For example, use either Fast Red orIN T/BCIP for red/brown precipitates.

!7 Cover each section with approximately200 µl color solution and incubate slidesin a humid chamber for 2–24 h in thedark.

!8 When color development is optimal, stopthe color reaction by incubating the slidesin buffer 3 [10 mM Tris-H Cl (pH 8.1), 1mM EDTA].

!9 Dip slides briefly in distilled water.

!2 Drain prehybridization buffer from theslides and overlay each section with 30 µl of hybridization buffer containing5–10 ng of digoxigenin-labeled RN Aprobe.

!3 Cover samples with a 24 x 30 mmhydrophobic plastic coverslip (e.g. cutfrom Gel Bond Film, FMC BioProducts,Rockland, ME, USA).

!4 Incubate sections at 42°C overnight in ahumid chamber.

Posthybridization

Caution: Use a separate set of glasswarefor posthybridization and prehybridizationprocedures to avoid RN ase contaminationin prehybridization steps.!1 Remove coverslips from sections by

immersing slides for 5–10 min in 2 x SSC.Caution: Do not place samples whichare hybridized w ith different probes inthe same container.

!2 In a shaking water bath at 37°C, washsections as follows:" 2 x15 min with 2 x SSC." 2 x15 min with 1x SSC.

!3 To digest any single-stranded (unbound)RN A probe, incubate sections for 30 minat 37°C in N TE buffer [500 mM N aCl,10 mM Tris, 1 mM EDTA, pH 8.0]containing 20 µg/ml RN ase A.Caution: Be particularly careful w ithRN ase. This enzyme is extremely stableand difficult to inactivate. Avoidcontamination of any equipment orglassware which might be used forprobe preparation or prehybridizationprocedures. Use separate glassware forRN ase-contaminated solutions.

!4 In a shaking water bath at 37°C, washsections 2 x 30 min with 0.1 x SSC.Note: I f this procedure gives high back -ground or nonspecific signals, try post-hybridization washings at 52°C with2 x SSC containing 50% formamide.

Immunological detection

Note: This detection procedure uses compo-nents of the DIG N ucleic Acid DetectionKit*. A lternative detection proceduresinclude the immunogold method with silverenhancement for enzyme-independentprobe detection (Komminoth et al. 1992;Komminoth et al., 1995) and other detec-tion procedures described in Chapter 5 ofthis manual.


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!3 Add a 24 x 30 mm coverslip to eachsection and incubate slides in a humidchamber at 37°C for 2 h.

!4 Remove coverslips by immersing slidesfor 5 min in 2xSSC.

In situ hybridization

Note: To increase the sensitiv ity of in situhybridization procedures, use mixtures ofoligonucleotide probes that are comple-mentary to different regions of the targetRN A.!1 Prepare hybridization buffer containing:

" 2 x SSC." 1 x Denhardt’s solution [0.02% Ficoll®,

0.02% polyvinylpyrrolidone, 10 mg/mlRN ase-free bovine serum albumin].

" 10% dextran sulfate." 50 mM phosphate buffer (pH 7.0)." 50 mM DTT." 250 µg/ml yeast t-RN A." 5 µg/ml polydeoxyadenylic acid." 100 µg/ml polyadenylic acid." 0.05 pM/ml Randomer O ligonucleo-

tide H ybridization Probe (DuPont)." 500 µg/ml denatured and sheared

salmon sperm DN A.Caution: Denature and add salmonsperm DN A to buffer shortly beforehybridization.

" Enough deionized formamide (% dF,as calculated by the formula below) toproduce stringent hybridization con-ditions at 37°C [that is, to make 37°C= Tm (oligonucleotide probe) – 10°C](Long et al., 1992).Caution: % dF should not exceed47% of the total volume of thehybridization buffer. I f dF is less than47% , add ddH 2O so that ddH 2O plusdF equals 47% of the volume.

Note: Use the following formula tocalculate the formamide concentration(% dF) in the hybridization buffer foreach oligonucleotide probe:

!10 Counterstain sections for 1–2 min with0.02% fast green FCF or 0.1% nuclearFast Red (Aldrich Chemical Corp.,Milwaukee, WI, USA) in distilled H 2O .

!11 Wash sections 2 x 10 min in tap water.!12 Mount sectionsusingan aqueousmounting

solution (e.g. Aqua-Mount from LernerLaboratories, Pittsburgh, PA, USA).Caution: Do not use xylene-basedmounting solutions. These lead to crystalformation of color precipitates.

IIIB. ISH protocol for detecting mRNAwithDIG-labeledoligonucleotideprobesProbe labeling!1 Label 100 pmol of oligonucleotide probe

(20–30 mer) by tailing with the DIGO ligonucleotide Tailing Kit* accordingto the protocol described in Chapter 4 ofthis manual.

!2 Purify labeled probes according to pro-cedures in the DIG/Genius™ SystemUser’s Guide for Membrane H ybridi-zation or in Chapter 4 of this manual.

!3 Estimate the yield of labeled probes bydirect blotting procedures as described inChapter 4 of this manual.

!4 Aliquot the labeled probes and storethem at –20°C.Note: Probes are stable for up to oneyear.

Note: To increase the sensitiv ity of in situhybridization procedures, prepare severallabeled probes that are complementary todifferent regions of the target RN A, thenuse mixtures of the probes for the hybridi-zation step below.

Prehybridization!1 Follow Steps 1–7 from the prehybridi-

zation section of Procedure IIIA (forRN A probes).

!2 O verlay each section with 40 µlprehybridization buffer (identical to thehybridization buffer to be used for in situhybridization below, but containing nolabeled probe).

(–7.9) + 81.5 + 0.41 (% GC) – 675/L – % mismatch – (47)% dF = _______________________________________________0.65

where, in this case: –7.9 = 16.6 log10 [N a+] (for 2 x SSC)% GC = % [G + C] base content of the oligonucleotideL = oligonucleotide length (in bases)% mismatch = % (bases in oligonucleotide not complementary to

target)47 = hybridization temperature + 10°C (for 37°C)


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N egative controls!1 N egative sample: Tissue or cell line

known to lack the sequence of interest.!2 Technical controls:

" Target: Digestion of mRN A withRN ase prior to in situ hybridization(should give negative results).

" H ybridization:– H ybridization with sense probe– H ybridization with irrelevant probe (e.g. probe for viral sequences)– H ybridization in the presence of ex-

cess unlabeled antisense probe(all should give negative results)." Detection:

– H ybridization without probe– O mission of anti-DIG antibody

(both should give negative results).

Results

Note: H ybridization buffer (minus dF,ddH 2O , and salmon sperm DN A) canbe stored at –20° C for several months.

!2 Drain 2 x SSC (Step 4, Prehybridization)from the slides and overlay each sectionwith 30 µl of hybridization buffercontaining 10–30 ng of digoxigenin-labeled oligonucleotide probe.

!3 Cover sections with a 24 x 30 mm hydro-phobic plastic coverslip (e.g. cut from GelBond Film, FMC BioProducts, Rockland,ME, USA).

!4 Incubate samples at 37°C overnight in ahumid chamber.

Posthybridization!1 Remove coverslips from sections by

immersing slides for 5–10 min in 2 x SSC.!2 In a shaking water bath at 37°C, wash

sections as follows:" 2 x 15 min with 2 x SSC." 2 x 15 min with 1x SSC." 2 x 15 min with 0.25 x SSC.

Immunological detection

Use the same immunological detectionprotocol as in Procedure IIIA (for RN Aprobes) above.

Controls for in situ hybridizationsAdequate controls must always be includedto ensure the specificity of detection signals.Controls should include positive andnegative samples as well as technical controlsto detect false positive and negative results(H errington and McGee, 1992).

Positive controls!1 Positive sample: Tissue or cell line known

to contain mRN A of interest.!2 Technical control to test quality of tissue

mRN A and the procedure reagents:Labeled poly(dT) probe (for oligonucleo-tide in situ hybridization only); labeledRN A or oligonucleotide probes comple-mentary to abundant “housekeepinggenes” (such as !-tubulin) to test qualityof tissue mRN A and the procedurereagents (should give positive results).

Figure 1: Comparison of 35S- and digoxigenin-labeled cRNA probes for the detection of seminal vesicle secretion protein II (SVS II) mRNA in cryostat sections of thedorsolateral rat prostate. Note the equal intensity of signals in acini of the lateral lobeobtained with isotopic (Panel A) and non-isotopic (Panel B) in situ hybridization procedures.Also note the absence of signal in the coagulating glands (arrows in Panel B) which serves asan internal negative control.

A B

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Figure 2: SVS II mRNA detection in contiguous glands of the ratprostate with a digoxigenin-labeled antisense RNA probe. Panel A: Strong signals are presentin epithelia of the ampullary glandand no signals are encountered in adjacent duct epithelia of the coagulating gland (arrow). Panel B: A higher magnificationshows that the hybridization signalis restricted to the cytoplasmic portion of the glandular epithelialcells (cryostat sections).

Figure 3: SVS II mRNA detection in contiguousglands of the rat prostate with a digoxigenin-labeled oligonucleotide antisense probe.Hybridization signals appear to be slightly lessstrong than with the SVS II RNA probe (Figure 2).

A

B

$

"

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Figure 4: Detection of parathyroid hormone(PTH) mRNA in formaldehyde-fixed, paraffin-embedded tissue of a parathyroid gland with a digoxigenin-labeled antisense RNA probe. Panel A: Note the weaker signal in cells of anadenomatous lesion (upper right part) comparedwith the normal parathyroid tissue. Panel B: Highermagnification shows the excellent resolution of hybridization signals, which are confined to thecytoplasmic portion of cells.

Figure 5: Use of a digoxigenin-labeled antisenseRNA probe and Fast Red chromogen to detectPTH mRNA in formaldehyde-fixed, paraffin-embedded tissue of a parathyroid gland withchief cell hyperplasia.Panel A: PTH mRNA is marked by red precipitates.Panel B: Only weak signals are present when asense PTH probe is used as a control.

Figure 6: Detection of synaptophysin mRNA informaldehyde-fixed, paraffin-embedded tissueof a neuroendocrine carcinoma of the gut withdigoxigenin-labeled oligonucleotide probes andimmunogold-silver enhancement method forvisualization.Note the strong hybridization signals (consisting ofsilver precipitates) over tumor cells in Panel A(where an antisense probe was used) and the muchweaker signal in Panel B (where the appropriatesense probe was used as a control).

A

A B

A B

B

"

"

"

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Reagent Description Cat. No. Pack size DIG RNA Labeling For RNA labeling with digoxigenin-UTP 1175 025 1 Kit (2x10 Kit*, **, *** by in vitro transcription with SP6 and labeling

T7 RNA polymerase. reactions)DIG Oligonucleotide For tailing oligonucleotides with 1417 231 1 Kit (25 tailingTailing Kit*, ***, **** digoxigenin-dUTP. reactions)DIG Nucleic Acid For detection of digoxigenin-labeled 1175 041 1 Kit Detection Kit* nucleic acids by an enzyme-linked

immunoassay with a highly specific anti-DIG-AP antibody conjugate and the color substrate NBT and BCIP.

Triton® X-100 Vicous, liquid 789 704 100 mlEDTA Powder 808 261 250 g

808 270 500 g808 288 1 kg

Proteinase K Solution 1413 783 1.25 ml1373 196 5 ml1373 200 25 ml

Pepsin Lyophilizate 108 057 1 g1693 387 5 g

BSA Highest quality, lyophilizate 238 031 1 g238 040 10 g

DTT Purity: > 97% 197 777 2 g708 984 10 g

1583 786 25 g708 992 50 g709 000 100 g

Tris Powder 127 434 100 g708 968 500 g708 976 1 kg

RNase A From bovine pancreas, dry powder 109 142 25 mg109 169 100 mg

tRNA From baker’s yeast, lyophilizate 109 495 100 mg109 509 500 mg

Poly(A) Polyadenylic acid 108 626 100 mgPoly(dA) Poly-deoxy-adenylic acid 223 581 5 A260 units

****Sold under the tradename of Genius in the US.****EP Patent 0 324 474 granted to Boehringer Mannheim GmbH.****Licensed by Institute Pasteur.****EP Patents 0 124 657/0 324 474 granted to Boehringer Mannheim GmbH.

ReferencesHerrington, C. S.; McGee, J. O. (1992) Principlesand basic methodology of DNA/RNA detection by in situ hybridization. In: Herrington, C. S.; McGee, J. O. (Eds.) Diagnostic molecular pathology: A practical approach. New York: IRL Press, Vol. 1,pp. 69–102.Komminoth, P. (1992) Digoxigenin as an alternativeprobe labeling for in situ hybridization. Diagn. Mol.Pathol. 1, 142–150.Komminoth, P.; Merk, F. B.; Leav, I.; Wolfe, H. J.;Roth, J. (1992) Comparison of 35S- and digoxigenin-labeled RNA and oligonucleotide probes for in situhybridization. Expression of mRNA of the seminalvesicle secretion protein II and androgen receptorgenes in the rat prostate. Histochemistry 98,217–228.Komminoth, P.; Roth, J.; Saremaslani, P.; Schrödel,S., Heitz, P. U. (1995) Overlapping expression ofimmunohistochemical markers and synaptophysinmRNA in pheochromocytomas and adrenocorticalcarcinomas. Implications for the differential diagnosis of adrenal gland tumors. Lab. Invest. 72,424–431. Long, A. A.; Mueller, J.; Andre-Schwartz, J.; Barrett,K.; Schwartz, R., Wolfe, H. J. (1992) High-specificityin situ hybridization: Methods and application.Diagn. Mol. Pathol. 1, 45–57.Naber, S.; Smith, L.; Wolfe, H. J. (1992) Role of thefrozen tissue bank in molecular pathology. Diagn.Mol. Pathol. 1, 73–79.Sambrook, J.; Fritsch, E.; Maniatis, T. (1989)Molecular Cloning: A Laboratory Manual. ColdSpring Harbor, NY: Cold Spring Harbor LaboratoryPress.Valentino, K. L.; Eberwine, J. H.; Barchas, J. D.(1987) In situ Hybridization: Applications ToNeurobiology. Oxford, England: Oxford UniversityPress.

Young, I. D.; Ailles, L.; Deugau, K.; Kisilevsky, R.(1991) Transcription of cRNA for in situ hybridizationfrom Polymerase Chain Reaction-amplified DNA.Lab. Invest. 64, 709–712.

AcknowledgmentsI am grateful to Ph. U. H eitz and J. Roth forgeneral support; to A. A. Long, H . J. Wolfe,S. P. N aber, and W. Membrino for technicaladvice; to M. Machado, X. Matias-Guiu, F.B. Merck, I. Leav, and P. Saremaslani fortechnical support; and to N . Wey and H .N ef for photographic reproductions. Theprojects – during which the protocols havebeen established – were supported in part bythe Julius Müller-Grocholski CancerResearch Foundation, Zürich Switzerland.

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*Soldunder the tradename of Geniusin the US.

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!4 Coat slides with a 2% solution of 3-aminopropyltriethoxy-silane (Sigma) inacetone, then air dry them.Caution: Perform Step 4 under RN ase-free conditions.

!5 Cut 4 µm thick sections from the fixedsamples with disposable knifes and attachthem to the prepared slides.Caution: Perform Step 5 under RN ase-free conditions.

!6 Dry the sections overnight at 55°C.!7 Store the sample slides at room temper-

ature until they are used.

III. Pretreatment of sectionsCaution: Perform pretreatment andhybridization steps under RN ase-freeconditions. Prepare all solutions andbuffers for these steps w ith DEPC-treatedwater (Sambrook et al., 1989). Bake allglassware for 4 h at 180°C. Assume thatdisposable plasticware is RN ase-free.!1 Dewax slides extensively by treating with

xylene (preferably overnight) and agraded series of ethanol solutions.

!2 To preserve the mRN A during thefollowing procedure, fix the sections onceagain with 4% paraformaldehyde (in100 mM phosphate buffer) for 20 min.

!3 Rinse the sections 3–5 times with TBSbuffer [50 mM Tris-H Cl (pH 7.5);150 mM N aCl].

!4 Treat the sections for 10 min with 200 mM H Cl to denature proteins.

!5 Rinse the sections 3–5 times with TBS.!6 To reduce nonspecific background (H a-

yashi et al., 1978), incubate the sectionsfor 10 min on a magnetic stirrer with afreshly mixed solution of 0.5% aceticanhydride in 100 mM Tris (pH 8.0).

!7 Rinse the sections 3–5 times with TBS.!8 Treat the sections for 20 min at 37°C with

Proteinase K (10–500 µg per ml in TBSwhich contains 2 mM CaCl2).

The concentration of Proteinase Kdepends upon the degree of fixation. Gen-erally, start with 20 µg/ml Proteinase K

The following protocol was developed toshow mRN A in paraffin embedded materialof the central nervous system. It also allowsdouble staining of protein and mRN A. Thismethod is sensitive enough to show mRN Aexpressed at very low levels (Breitschopf etal., 1992).

I. Probe preparation!1 Prepare plasmids by standard methods

(Sambrook et al., 1989).!2 Prepare RN A probes from the plasmids

and label them with digoxigenin (DIG)either according to the methods inChapter 4 of this manual or according tothe instructions in the DIG RN ALabeling Kit*.

!3 Remove unincorporated nucleotidesfrom the labeled probes by passing thelabeling mixture over a Q uick Spin™column for radiolabeled RN A prepara-tion (Sephadex® G-50).Caution: O mission of this column stepmay lead to background problems.

!4 Determine the amount of DIG-labelingwith a dot blot according to the methodin Chapter 4 of this manual.

II. Tissue preparation!1 Use samples fixed by standardized

methods, such as:" Perfusion-fixed samples.

Caution: Before using perfusion-fixedsamples, treat them further by immer-sion-fixing for 3–4 h at room tem-perature in 100 mM phosphate buffercontaining 4% paraformaldehyde.Note: Samples fixed with 4% para-formaldehyde give the best results inthis procedure.

" Routinely fixed autopsy samples." Samples fixed with 2.5% glutar-

aldehyde.!2 After fixation, wash the samples in

phosphate buffered saline (PBS).!3 Embed the fixed samples in paraffin.

Detection of mRNA on paraffin embedded material of the central nervous system with DIG-labeled RNA probesH . Breitschopf and G . Suchanek , Research Unit for Experimental N europathology, Austrian Academy of Sciences, V ienna, Austria.

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" 0.01% sheared salmon sperm DN A(Sambrook et al., 1989).

" 0.02% SDS." 50% formamide.Caution: The concentration of dextransulfate is critical. Without appropriateamounts of dextran sulfate, the methodloses sensitiv ity. H owever, excessiveconcentrations of dextran sulfate causeshigher v iscosity of the hybridizationbuffer. To prevent uneven distribution ofthe probe and uneven signals, alwaysvor-tex the hybridization buffer extensively.

!3 Dilute the labeled antisense RN A probeto an appropriate degree in hybridizationbuffer. The diluted probe solution maycontain as much as 1 part labeled probe to4 parts hybridization buffer.Note: The amount of probe neededdepends upon the degree of probelabeling (as determined in Procedure I ,Step 4 above). Generally, use the lowestprobe concentration that gives optimalresponse in that dot blot procedure.Example: In a serial dilution of theprobe on a dot blot , if a 1:160 dilutiongives a significantly stronger responsethan a 1:320 dilution, perform the initialhybridization experiments w ith both a1:200 and a 1:300 dilution of the probe.

!4 Pipette the diluted antisense probesolution onto each section at a volume of 10 µl/cm2. Cover with a coverslip.Note: In our experience, a prehybridi-zation step (w ith hybridization bufferalone) did not improve results.

!5 As a control serve sections treated in thesame way whereby in steps 3 and 4 a labeled sense RN A probe is used.Caution: Control hybridization w ithsense probes is necessary to prove thespecificity of the reaction. H owever,hybridization w ith sense probes some-times gives unexpected hybridizationsignals or background staining.

!6 Place the slides on a hot plate at 95°C for4 min.Note: This step increases the signal fromRN A/RN A hybrids.

!7 Incubate the slides in a humid chamberfor 4–6 h at 55°–75°C.Note: H ybridization specificity can beincreased by increasing the temperatureof hybridization, if necessary, as high as75°C. Increasing the temperature canhelp to differentiate mRN As of highlyhomologous proteins (Taylor et al.,1994).

for paraformaldehyde-fixed tissue. Theconcentration needed for glutaraldehyde-fixed or overfixed tissue is generallyhigher than for paraformaldehyde-fixedtissue.Caution: Determine the optimal con-centration of Proteinase K empiricallyfor each type of fixation. O ne of themost critical steps in the whole proce-dure is finding the balance betweenprior fixation and the proper concen-tration of Proteinase K. Proteinase Kconcentrations which are too low or toohigh may lead to false negative results.(See Figure 3 under “Results”.) O urexperience shows that autolysis causesfewer problems than overfixation does.Even after 30 hours autolysis, we coulddemonstrate mRN A. (See Figure 4 under“Results”.) In overfixed material, wewere not always successful in demon-strating mRN A, even when we increasedProteinase K to very high concentrations(up to 500 mg/ml).

!9 Rinse the sections 3–5 times with TBS.!10 Incubate the sections at 4°C for 5 min

with TBS (pH 7.5) to stop the ProteinaseK digestion.Caution: Do not postfix the sectionswith paraformaldehyde after proteasedigestion, since this reduces signal intensity.

!11 Dehydrate the sections in a graded seriesof ethanol solutions (increasing concen-trations of ethanol).

!12 Rinse the sections briefly withchloroform.Note: At this point, you may, if necessary,store the sections under dust- andRN ase-free conditions for several days.

IV. HybridizationCaution: Perform the hybridization stepunder RN ase-free conditions. Prepare allsolutions and buffers for this step w ithDEPC-treated water (Sambrook et al.,1989). Bake all glassware for 4 h at 180°C.Assume that disposable plasticware isRN ase-free.!1 Place the sections in a humid chamber at

55°C for 30 min.!2 Prepare a hybridization buffer by mixing

the following components, then vor-texing vigorously:" 2 x SSC (Sambrook et al., 1989)." 10% dextran sulfate.

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!1 Incubate slide overnight at 4°C withprimary antibody (either polyclonal ormouse monoclonal), diluted as necessaryin TBS containing 10% fetal calf serum(TBS-FCS).Example: The polyclonal antibody forproteolipid protein used in Figure 1 wasdiluted 1:1000.

!2 Rinse the slide 3–5 times with TBS atroom temperature.Note: Perform Step 2 as well as all theremaining incubation and wash steps ofProcedure VI at room temperature.

!3 Depending upon the type of primaryantibody used in Step 1, do one of thefollowing:" I f the primary antibody was a poly-

clonal antibody from rabbit, incubatethe slide for 60 min with anti-rabbitserum from mouse [Dakopatts], dil-uted 1:100 in TBS-FCS, then rinse theslide 3–5 times with TBS. Go to Step 4.

" I f the primary antibody was a mousemonoclonal antibody, skip this stepand go to Step 4.

!4 Incubate slide for 60 min with anti-mouseserum (from rabbit [Dakopatts], diluted1:100 in TBS-FCS), then rinse the slide3–5 times with TBS.

!5 Incubate slide for 60 min with APAAPcomplex (mouse) (diluted 1:100 in TBS-FCS), then rinse the slide 3–5 times withTBS.

!6 Incubate slide for 30 min with anti-mouseserum (from rabbit) (diluted 1:100 inTBS-FCS), then rinse the slide 3–5 timeswith TBS.

!7 Incubate slide for 30 min with APAAPcomplex (mouse) (diluted 1:100 in TBS-FCS), then rinse the slide 3–5 times withTBS.

!8 Repeat Steps 6 and 7.Note: Repeating the APAAP steps w illenhance the signal.

!9 Prepare APAAP substrate by dissolving200 mg naphthol-ASMX-phosphate,20 ml dimethylformamide, and 1 ml of1 M levamisole (all from Sigma) in 980 mlTris-H Cl (pH 8.2).

!10 Dissolve 50 mg Fast Red TR Salt in 50 mlof APAAP substrate, then filter thesolution.

!11 In a Coplin jar at room temperature,incubate the slides with the Fast Red-APAAP substrate solution until the colordevelops.

V. Washes and detection of mRNANote: Stringency washings, often describedas a method to remove nonspecificallybound probes, are helpful in reducingdiffuse background staining, but do notsignificantly improve the specificity of thehybridization.!1 Incubate the slides in 2 x SSC overnight.

Note: The coverslips will float off duringthis incubation.

!2 Wash the slides as follows:" 3 x 20 min at 55°C with 50% deionized

formamide in 1x SSC." 2 x15 min at room temperature with

1x SSC .!3 Rinse the sections 3–5 times with TBS.!4 Incubate the slides for 15 min with

blocking mixture (blocking reagentcontaining 10% fetal calf serum).

!5 Incubate the slides for 60 min withalkaline-phosphatase-conjugated anti-DIG antibody [diluted 1:500 in blockingmixture (from Step 4 above)].

!6 Rinse the sections 3–5 times with TBS.!7 Prepare N BT/BCIP color reagent as rec-

ommended by the manufacturer.!8 In a Coplin jar in the refrigerator,

incubate the sections with color reagentuntil sufficient color develops.Note: Incubation can be extended up to120 h if the color reagent is replacedevery time it precipitates or changes color.

!9 Stop the color reaction by rinsing theslides several times with tap water.

!10 Finally, rinse the slides with distilledwater.

!11 Mount the slides directly with any water-soluble mounting medium.Note:O ptionally, counterstain the slidesor use immunocytochemistry (as in Pro-cedure VI below) to v isualize proteinson the slides.

VI. Detection of proteins by immunocytochemistryNote: This protocol describes a tripleAPAAP (alkaline phosphatase anti-alka-line phosphatase) reaction (Vass et al.,1989), which allows v isualization ofproteins on the same slide with the mRN A.See Figure 1 under “Results” for anexample of double staining obtained w iththis technique.

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Note: I f you substitute PAP complex forAPAAP complex, modify the aboveprocedure as follows:

" Perform all rinses and all dilutions w ithPBS rather than TBS.

" For the color reaction, use filtered dia-minobenz idine reagent (50 mg diamino-benz idine dissolved in 100 ml PBSwhich contains 0.01% H 2O 2)

For an example of immunostaining w ithPAP complex, see Figure 2 under “Results”.

Results

Figure 1: Double staining of mRNA and protein(APAAP method) in a normal rat brain. The mRNA (black-blue) for proteolipid protein (PLP) wasdetected by the procedure described in the text andstained with NBT/BCIP color reagent. Proteolipidprotein (red) was detected with a 1:1000-dilutedrabbit polyclonal antibody and visualized withAPAAP and Fast Red TR Salt.

Figure 2: Double staining of mRNA and protein (PAP method) in a normal rat brain.The experiment is identical to that in Figure 1,except PLP (brown) was visualized with PAP anddiaminobenzidine reagent.

Figure 3: Effect of paraformaldehyde and glutaraldehydefixation on in situ hybridization of mRNA. PLP mRNA was detected in sections of rat spinal cord which had been fixedbydifferent methods. Panel a: Sectionswerefixed with 4% para-formaldehyde and then treated with different concentrations ofProteinaseK. Fromtop tobottomtheProteinaseKconcentrationswere: 0.002%, 0.005%, 0.02%, 0.05%. Panel b: Sections werefixed in 2.5% glutaraldehyde, then treated with the sameconcentrations of Proteinase K as in Panel a.

a b

PFA GA

0.002%

0.005%

0.02%

0.05%

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AcknowledgmentsThe rabbit polyclonal antibody againstproteolipid protein (PLP) was a gift from Dr. S. Piddlesden, University of Cardiff, UK.

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Figure 4: Effect of autolysis andoverfixation on in situ hybridiza-tion of mRNA and immunocyto-chemical staining. In spite ofpronounced autolytic changes thatoccur postmortem, a good PLPmRNA signal can be seen in humanbrain autopsy samples (Panel a).Although in well preserved and fixedexperimental tissue (Panel b) both,the signal for in situ hybridization aswell as the signal for immunocyto-chemistry is much more distinct.Protein (red) and mRNA (blue-black)were stained as in Figure 1.

a

b

ReferencesBreitschopf, H; Suchanek, G; Gould, R. M.; Colman, D. R.; Lassmann, H. (1992) In situhybridization with digoxigenin-labeled probes: Sensitive and reliable detection method applied to myelinating rat brain. Acta Neuropathol. 84,581–587.Hayashi, S; Gillam, I. C.; Delaney, A. D.; Tener, G. M. (1978) Acetylation of chromosomesquashes of Drosophila melanogaster decreasesthe background in autoradiographs fromhybridization with 125I-labeled RNA. J. Histochem.Cytochem. 26, 677–679.Sambrook, J; Fritsch, E. F.; Maniatis, T. (1989)Molecular cloning: A Laboratory Manual, 2nd ed.Cold Spring Harbor, N.Y.: Cold Spring HarborLaboratory Press.Taylor, V.; Miescher, G. C.; Pfarr, S.; Honegger, P.;Breitschopf, H.; Lassmann, H.; Steck, A. J. (1994)Expression and developmental regulation of Ehk-1,aneuronal Elk-likereceptor tyrosine kinase in brain.Neuroscience 63, 163–178.Vass, K.; Berger, M. L.; Nowak, T. S. Jr.; Welch, W. J.; Lassmann, H. (1989) Induction of stressprotein HSP 70 in nerve cells after statusepilepticus in the rat. Neurosci. Lett. 100, 259–264.



T7 RNA polymerase. reactions)Proteinase K Lyophilizate 161 519 25 mg

745 723 100 mg1000 144 500 mg1092 766 1 g

Tris Powder 127 434 100 g708 968 500 g708 976 1 kg

Blocking Reagent Powder 1096 176 50 gAnti-DIG-AP Fab Fragments from sheep 1093 274 150 U (200 µl)NBT/BCIP Stock solution 1681 451 8 ml

"

***Sold under the tradename of Genius in the US.***EP Patent 0 324 474 granted to Boehringer Mannheim GmbH.***Licensed by Institute Pasteur.

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I. Fixation, dehydration, and embedding

Follow the protocol described by Jackson(1992) to fix, dehydrate, and embed thetissue in paraffin, with the followingmodifications:!1 For fixation, prepare either of the follow-

ing solutions:" 100 mM phosphate buffer, pH 7,

containing 0.25% gluteraldehyde and4% freshly depolymerized parafor-maldehyde.

" Formalin-acetic acid (50% ethanol;10% formalin, containing 37% form-aldehyde; 5% acetic acid).

!2 Using either of the solutions from Step 1, fix the tissue for 4 h at roomtemperature. During the fixation:" Vacuum infiltrate the tissue (with a

water aspirator) for 10 min once anhour.

" After each vacuum infiltration, renewthe fixative solution.

!3 Depending on the fixative used in Step 2, wash the fixed tissue as follows:" I f gluteraldehyde-paraformaldehyde

was the fixative, wash the fixed tissue2 x 30 min with 100 mM phosphatebuffer, pH 7.

" I f formalin-acetic acid was the fixative,wash the fixed tissue 2 x 30 min with50% ethanol.

!4 Dehydrate the tissue by incubating in thefollowing series of ethanol solutions:" Either 90 min (after gluteraldehyde-

paraformaldehyde fixation) or 30 min(after formalin-acetic acid fixation) atroom temperature in 50% ethanol.

" 90 min at room temperature in 70%ethanol.

" O vernight at 4°C in 85% ethanol." 3 x 90 min at room temperature in

100% ethanol.

Note: The following article is reprintedwith the permission of Academic Press(Copyright® 1993 by Academic Press, Inc.)and H arcourt Brace & Company and isbased on the original article: DeBlock , M.,Debrouwer, D . (1993) RN A-RN A in situhybridization using digoxigenin-labeledprobes: the use of high molecular weightpolyv inyl alcohol in the alkaline phospha-tase indoxyl-nitroblue tetrazolium. AnalyticalBiochemistry 216; 88–89.

The indoxyl-nitroblue tetrazolium (BCIP-N BT) reaction is relatively slow. During thereaction, intermediates (indoxyls) diffuseaway into the medium, making it difficult tolocalize the site of hybridization (VanN oorden and Jonges, 1987) and reducing thehybridization signal.

In this paper, an improved nonradioactiveRN A-RN A in situ hybridization protocolusing alkaline phosphatase-conjugated dig-oxigenin- (DIG-) labeled probes is presented.The addition of polyvinyl alcohol (PVA) ofhigh molecular weight (40–100 kD) to theBCIP-N BT detection system enhances thealkaline phosphatase reaction and preventsdiffusion of reaction intermediates, resultingin a twentyfold increase in sensitivity withoutincreasing the background. Due to the morelocalized precipitation of the formazan, thesite of hybridization can be determined moreprecisely.

RNA-RNA in situhybridization using DIG-labeledprobes: the effect of high molecular weight polyvinyl alcohol on the alkaline phosphatase indoxyl-nitroblue tetrazolium reactionMarc DeBlock and Dirk Debrouwer, Plant Genetic Systems N .V., Gent, Belgium.

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Figure 1: The influence of PVA on the alkaline phosphatase BCIP-NBT reaction in in situhybridizations. The hybridizations were carried out on 10 µm paraffin sections of stigmas and styles oftobacco with antisense and sense DIG-labeled RNA probes of pMG07 (de S. Goldman et al., 1992). C, cortex tissue; TT, transmitting tissue; V, vascular tissue. Bars = 200 µm. Panel a: Hybridization with antisense probe. Twenty percent 10kDPVA was added to the alkaline phosphatase reaction buffer. Development was done for 4 h. Panel b: Hybridization with antisense probe. Ten percent 70–100 kD PVA was added to the alkalinephosphatase reaction buffer. Development was done for 2 h. Panel c: Hybridization with sense RNA probe. Ten percent 70–100 kD PVA was added to the alkalinephosphatase reaction buffer. Development was done for 20 h.

a b c

Table 1: Influence of polymers on the alkaline phosphatase BCIP-NBT reactiona.

a Each reaction was done with a total volume of 2 ml BCIP-NBT reaction mixture in a test tube. The BCIP-NBT reaction mixture was as described in the text, except that it contained 7.5 X 10–3 units alkaline phosphatase/ml and the polymer indicated in the table. Incubation was for 30 min at 24°C.

b The polymers were dissolved in the alkaline phosphatase reaction buffer in concentrations between 10%and 30%. The concentrations are given as a percentage of weight to volume.

c A solution of 30% 10 kD PVA was too viscous.d Solutions of 20 or 30% 40 kD PVA and 20 or 30% 70–100 kD PVA were too viscous.e The amount of formazan formed was measured at 605 nm (Eadie et al., 1970). The enhancement factor

is expressed with respect to the controls (no polymer added). If no polymer was added, about 0.01 mMformazan was formed.

Polymer Molecular Concentrationb Effect on the alkalineweight phosphatase BCIP-N BT reactione

PVP 15 kD 10, 20, 30% Autoreduction of N BT25 kD 10, 20, 30%44 kD 10, 20, 30%

PEG 6 kD 10, 20, 30% N o enhancement 20 kD 10, 20, 30% (0.01 mM formazan)

PVA 10 kD 10, 20% c Approx. 4-fold enhancement(0.04 mM formazan)

40 kD 10% d Approximately 6- to 8-fold enhance-70–100 kD 10% d ment (0.06 to 0.08 mM formazan)

#

#

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ReferencesDeBlock, M.; Debrouwer, D. (1993) RNA-RNA in situhybridization using digoxigenin-labeled probes: the use of high molecular weight polyvinyl alcoholin the alkaline phosphatase indoxyl-nitrobluetetrazolium reaction. Anal. Biochem. 215, 86–89.de S. Goldman, M. H.; Pezzotti, M.; Seurinck, J.;Mariani, C. (1992) Developmental expression oftobacco pistil-specific genes encoding novelextensin-like proteins. Plant Cell 4, 1041–1051.Eadie, M. J.; Tyrer, J. H.; Kukums, J. R.; Hooper, W. D. (1970) Histochemie 21, 170–180.Jackson, D. (1991) In situ hybridization in plants. In: Gurr, S. J.; McPherson; Bowles, D. J. (Eds.)Molecular Plant Pathology, A Practical Approach.Oxford, England: Oxford University Press, Vol. 1, pp. 163–174.Van Noorden, C. J. F.; Jonges, G. N. (1987) Quantification of the histochemical reaction for alkaline phosphatase activity using the indoxyl-tetranitro BT method. Histochem. J. 19, 94–102.



T7 RNA polymerase. reactions)Proteinase K Lyophilizate 161 519 25 mg

745 723 100 mg1000 144 500 mg1092 766 1 g

tRNA From baker’s yeast, lyophilizate 109 495 100 mg109 509 500 mg

DNA from herring Lyophilized, sodium salt 223 646 1 gspermDTT Purity: > 97% 197 777 2 g

708 984 10 g1583 786 25 g

708 992 50 g709 000 100 g

RNase A Powder 109 142 25 mg109 169 100 mg

RNase Inhibitor From human placenta 799 017 2,000 units799 025 10,000 units

DIG Nucleic Acid For detection of digoxigenin-labeled 1175 041 1 Kit Detection Kit* nucleic acids by an enzyme-linked

immunoassay with a highly specific anti-DIG-AP antibody conjugate and the color substrates NBT and BCIP.


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" Incubate 4–16 h at –20°C." Centrifuge in a microcentrifuge for 15

min at 4°C to pellet the RN A." Discard the supernatant and dry the

RN A pellet in a vacuum desiccator." Resuspend labeled probe RN A in

DEPC-treated water at 10–50 µg/ml.!4 Prepare a hybridization mixture con-

taining the following:" 50% deionized formamide." 2.25 x SSPE (300 mM N aCl; 20 mM

N aH 2PO 4; 2 mM EDTA; pH 7.4)." 10% dextran sulfate." 2.5 x Denhardt’s solution." 100 µg/ml sheared and denatured

herring sperm DN A." 100 µg/ml tRN A." 5 mM DTT." 40 units/ml RN ase inhibitor." 0.2–1.0 µg/ml hydrolyzed and dena-

tured probe (200 bases long).!5 Cover each section with 250–500 µl of

hybridization mixture (depending on thesize of the section) and incubate in ahumidified box at 42°C overnight.Note: Do not use a coverslip during thehybridization incubation.

!6 After hybridization, wash the slides asfollows:" 5 min at room temperature with

3 x SSC.Note: 1x SSC contains150 mM N aCl,15 mM N a-citrate; pH 7.

" 5 min at room temperature with N TE(500 mM N aCl, 10 mM Tris-H Cl, 1 mM EDTA; pH 7.5).

!7 To remove unhybridized single-strandedRN A probe, put slides into a humidifiedbox and cover each section with 500 µl of N TE buffer containing 50 µg/ml RN ase A. Incubate for 30 min at37°C.

!8 After RN ase treatment, wash the slides3 x 5 min at room temperature with N TE.

!9 To remove nonspecifically hybridizedprobe, wash the slides as follows:" 30 min at room temperature with

2 x SSC." 1 h at 57°C with 0.1 x SSC.

II. Sectioning!1 Cut the paraffin-embedded tissues in

10 µm sections.!2 Attach the sections at 75°C for 1 h to

slides that have been treated with Vecta-bond (Vector Laboratories, Burlingame,CA, U.S.A.).

III. Prehybridization treatments!1 Dewax and hydrate the sections as

previously described (Jackson, 1992).!2 Incubate the sections with a Proteinase K

solution (100 mM Tris, pH 7.5;50 mM EDTA; 2 µg/ml Proteinase K) for30 min at 37°C.

!3 After the Proteinase K treatment, washthe slides 2 x in phosphate buffered saline(PBS).

!4 Dehydrate the sections in ascendingconcentrations of ethanol as previouslydescribed (Jackson, 1991).

IV. Hybridization!1 Label the probe RN A with DIG-UTP

according to the procedures given inChapter 4 of this manual.

!2 Reduce the length of the probe toapproximately 200 bases as follows:" To 50 µl of labeled probe RN A in

a microcentrifuge tube, add 30 µl 200 mM N a2CO 3 and 20 µl 200 mMN aH CO 3.

" H ydrolyze the probe at 60°C for tmin, where:t = (L0 – Lf) / (K · L0 · Lf)

L0 = starting length of probe RN A (in kb)

Lf = length of probe RN A (in kb) (In this case, Lf = 0.2 kb.)

K = rate constant (In this case, K = 0.11 kb/min.)

t = hydrolysis time in min!3 After hydrolysis, purify the probe RN A

as follows:" Add the following to the hydrolyzed

probe solution:– 5 µl 10% acetic acid– 11 µl 3 M sodium acetate (pH 6.0)– 1 µl of a 10 mg/ml tRN A stock– 1.2 µl 1 M MgCl2– 300 µl (about 2.5 volumes) cold

ethanol

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!6 Dehydrate the sections by incubating theslides without shaking in the followingethanol solutions:" 15 s in 70% ethanol." 2 x15 s in 100% ethanol.

!7 Air dry the slides and mount them withEukitt (O . Kindler GmbH , FRG).

!8 Examine the sections with a Dialux 22 microscope (Leitz, Wetzlar, FRG)equipped with N ormaski differential interference contrast.

Results and discussionThe direct influence of the polymers on theBCIP-N BT alkaline phosphatase reaction ina test tube is shown in Table 1. From theseresults it is clear that polyvinyl alcohol wasthe only polymer that enhanced formazanformation in the alkaline phosphatasereaction.

This enhancement was even more pro-nounced in in situ hybridization experi-ments. Figure 1 shows the results of an in situ hybridization in which sections of tobacco pistils were hybridized with anantisense RN A probe from a pistil-specificcDN A clone (de S. Goldman et al., 1992).After three hours development no hybridi-zation could be detected if no PVA had beenadded to the reaction buffer. A clear but stillweak signal was visible after three h if 20%PVA (molecular weight, 10 kD) was used(Figure 1a). H owever, with 10% PVA(molecular weight, 70–100 kD), a stronghybridization signal appeared in thetransmitting tissue of the pistil after a few h(Figure 1b). In the control with the senseRN A probe no signal could be detected,even after 24 h of development (Figure 1c).

V. Detection of DIG-labeled hybrids!1 Incubate the slides first with blocking

solution, then with blocking solutioncontaining 1.25 units/ml of alkalinephosphatase-conjugated anti-DIG Fabfragments as recommended in the packinsert of the DIG N ucleic Acid DetectionKit*.

!2 After the antibody incubation, wash theslides to remove unbound antibody asrecommended in the BoehringerMannheim DIG/Genius™ System User’sGuide for Membrane H ybridization.

!3 Prepare the BCIP-N BT-PVA color devel-opment solution as follows:" Prepare a Tris-N aCl-PVA stock solu-

tion by dissolving 10% (w/v) poly-vinyl alcohol [PVA, either 40 kD or 70–100 kD (Sigma)] at 90°C in 100 mM Tris-H Cl (pH 9) containing100 mM N aCl.

" Cool the Tris-N aCl-PVA stock solu-tion to room temperature.

" Add MgCl2, BCIP, and N BT to theTris-N aCl-PVA stock to produce afinal color development solution thatcontains:– 5 mM MgCl– 0.2 mM 5-bromo-4-chloro-3-indolyl

phosphate (BCIP)– 0.2 mM nitroblue tetrazolium salt

(N BT).!4 After the washes in Step 2, perform the

visualization step as follows:" Place the slides in 30 ml of BCIP-

N BT-PVA color development solutionin a vertical staining dish suited foreight slides.

" Incubate the slides in the color devel-opment solution for 2–16 h at 30°C.

" Monitor color formation visually.!5 When the color on each slide is optimal,

stop the color reaction by washing theslide 3 x 5 min in distilled water.

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Detection of neuropeptide mRNAs in tissue sections using oligonucleotides tailed with fluorescein-12-dUTP or DIG-dUTPDepartment of Cytochemistry and Cytometry, University of Leiden, The N etherlands.

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!1 Perform the hybridization as follows:" Prepare hybridization mixture [25%

formamide; 3 x SSC; 0.1% polyvinyl-pyrrolidone; 0.1% ficoll, 1% bovineserum albumin; 500 µg/ml shearedsalmon sperm DN A; 500 µg/ml yeastRN A].Note: 1x SSC contains 150 mM sodiumchloride, 15 mM sodium citrate; pH 7.0.

" To the cryostat sections, add hybridi-zation mixture containing 1 ng/µllabeled probe.

" Incubate for 2 h at room temperature.Note: I f the probe contains a fluoro-chrome label, perform the hybridiza-tion incubation in the dark .

!2 After hybridization, do the following:" Rinse the section 3 times in 4 x SSC at

room temperature." Dehydrate the sections.

Note: I f the probe contains a fluoro-chrome label, perform the washes inthe dark .

IV. Hybrid detectionDepending on the label used on the probe,follow one of these procedures:" If sections are hybridized with fluorescein-

labeled oligonucleotides:" Mount sections in PBS/glycerol (1:9;

v/v) containing 2.3% 1,4-diazabicyclo-(2,2,2)-octane (DABCO , from Sigma)and 0.1 µg/µl 4' ,6'-diamidino-2-phenylindole (DAPI).

" Evaluate under a fluorescence micro-scope.

" I f sections are hybridized with DIG-labeled oligonucleotides:" Make a 1:250 dilution of fluorescein-

conjugated anti-DIG antibody (fromsheep) in incubation buffer [100 mMTris-H Cl, pH 7.4; 150 mM N aCl; 1%blocking reagent].

" O verlay the sections with the dilutedantibody in incubation buffer.

" Incubate sections at room temperaturefor 30 min.

The protocol given below has been devel-oped with the neuropeptidergic system ofthe pond snail Lymnaea stagnalis. Moreinformation concerning the application andin situ hybridization methodology can befound in Van Minnen et al. (1989), Dirks etal. (1988), Dirks et al. (1990), and Dirks et al.(1991).

I. Tissue preparation!1 Dissect the tissue, embed in O .C.T.

compound (Miles Scientific, USA), andfreeze in liquid nitrogen.

!2 Prepare sections as follows:" Cut cryostat sections of 8 µm." Mount on poly-L-lysine-coated slides." Air dry.

!3 After mounting, do the following:" Fix the sections for 30 min at 4°C with

modified Carnoy’s [2% formalin,(from 37% stock), 75% ethanol, 23%acetic acid].

" Rinse the slides with water." Dehydrate the sections.

II. Probe preparationSynthesize and purify the oligonucleotidesaccording to routine procedures. Tail theoligonucleotide with DIG-dUTP or fluores-cein-dUTP according to the proceduresgiven in Chapter 4, but without using dATP.

Note: I f you use fluorescein-dUTP, add tothe labeling mixture equal amounts ofunmodified dTTP and fluorescein-dUTP,then carry out the labeling procedure inthe dark .

III. In situ hybridizationNote: 18-mers are used in this study. Foroligonucleotides w ith other lengths and/or composition, you may have to alter thestringency of hybridization by changingthe formamide concentration or the hy-bridization temperature listed below.

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***Sold under the tradename ofGenius in the US.

***EP Patents 0 124 657/0 324 474granted to Boehringer MannheimGmbH.


Dirks, R. W.; Raap, A. K.; Van Minnen, J.;Vreugdenhil, E.; Smit, A. B.; Van der Ploeg, M.(1989) Detection of mRNA molecules coding forneuropeptide hormones of the pond snail Lymnaeaestagnalis by radioactive and nonradioactive in situhybridization: a model study for mRNA detection. J. Histochem. Cytochem. 37, 7–14.Van Minnen, J.; Van de Haar, Ch.; Raap, A. K.;Vreugdenhil, E. (1988) Localization of ovulationhormone-like neuropeptide in the central nervoussystem of the snail Lymnaeae stagnalis by means ofimmunocytochemistry and in situ hybridization. Cell Tissue Res. 251, 477–484.


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ReferencesDirks, R. W.; van Gijlswijk, R. P. M.; Vooijs, M. A.;Smit, A. B.; Bogerd, J.; Van Minnen, J.; Raap, A. K.;Van der Ploeg, M. (1991) 3'-end fluorochromizedand haptenized oligonucleotides as in situ hybridi-zation probes for multiple, simultaneous RNAdetection. Exp. Cell Res. 194, 310–315.Dirks, R. W.; van Gijlswijk, R. P. M.; Tullis, R. H.;Smit, A. B.; Van Minnen, J.; Van der Ploeg, M.;Raap, A. K. (1990) Simultaneous detection ofdifferent mRNA sequences coding for neuropeptidehormones by double in situ hybridization using FITC-and biotin-labeled oligonucleotides. J. Histochem.Cytochem. 38, 467–473.

" Rinse sections 3 x 5 min in Tris-N aCl[100 mM Tris, pH 7.4;150 mM N aCl].

" Mount and evaluate as for fluorescein-labeled oligonucleotides.

Results

Figure 1: Direct detection of caudodorsal cellswith fluorescein-labeled CDCH-I.

Figure 2: Indirect detection ofcaudodorsal cellswithDIG-labeledCDCH-I and sheep anti-DIG-fluo-resceinconjugate. Thesamecellsare positive, but the hybridizationsignal is clearly more intense thanthat obtainedwith thedirect tech-nique(Figure1).

Reagent Description Cat. No. Pack size DIG Oligonucleotide For tailing oligonucleotides with 1417 231 1 Kit (25 tailingTailing Kit*, **, *** digoxigenin-dUTP. reactions)Fluorescein-12-dUTP Tetralithium salt, solution 1373 242 25 nmol (25 µl)tRNA From baker’s yeast, lyophilizate 109 495 100 mg

109 509 500 mgAnti-Digoxigenin- Fab Fragments from sheep 1207741 200 µg FluoresceinAnti-Digoxigenin- Fab Fragments from sheep 1207750 200 µg RhodamineDAPI Fluorescence dye for staining of 236 276 10 mg

chromosomesBlocking Reagent Blocking reagent for nucleic acid 1096 176 50 g

hybridizationBSA Highest quality, lyophilizate 238 031 1 g

238 040 10 g

#

$

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In situ hybridization of DIG-labeled rRNA probes to mouse liver ultrathin sectionsDr. D . Fischer, D . Weisenberger, and Prof. Dr. U . Scheer, Institute for Zoology I , University of Würzburg, Germany.

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!4 Transfer the liver cubes to other petridishes on ice, containing the fixatives (i)4% PFA and (ii) 0.5% GA plus 4% PFA.

!5 Incubate the cubes in the fixatives forabout 2 h.

!6 To remove the fixative, wash the cubes 3 x 5 min with ice cold PBS.

IB. Dehydration of materialTransfer the object cubes to preparativeglasses and dehydrate them in a series ofethanol solutions according to the followingscheme:" 2 x15 min in 30% ethanol at 4°C." 1x 30 min in 50% ethanol at 4°C." 1x 30 min in 50% ethanol at –20°C." 2 x 30 min in 70% ethanol at –20°C." 2 x 30 min in 90% ethanol at –20°C." 2 x 30 min in 96% ethanol at –20°C." 2 x 30 min in 100% ethanol at –20°C.

Caution: Leave the objects in a smallamount of liquid during changes from oneethanol solution to another. This preventsthem from drying out, especially at higherethanol concentrations.

IC. InfiltrationAfter dehydration, ethanol must be infiltratedwith the embedding medium Lowicryl K4M(Chemische Werke Lowi, Waldkraiburg,FRG). This resin tolerates a little residual waterin the object and produces good results inimmunocytochemistry.

Caution: When work ing w ith LowicrylK4M, perform all steps:– At low temperatures– Under the fume hood – While wearing gloves– While excluding air (oxygen) from K4M as

much as possible.

To exclude air from the embedding medium,do one of the following:" Fill all vessels which contain K4M with

nitrogen." Perform all steps in a closed chamber

cooled to constant temperature by liquidnitrogen, thus providing a nitrogen-satu-rated atmosphere in the closed prepa-ration chamber (e.g., “CS-auto”, Reichert& Jung, Cambridge Instr.).

Thin section electron microscopy of nucleolifrom mammalian cells reveals threenucleolar substructures. These are thefibrillar centers (FC), the dense fibrillarcomponent, which forms a closely apposedlayer upon the FCs, and the granularcomponent, which constitutes the bulk ofthe nucleolar mass. Although there is nodoubt that this ultrastructural organizationsomehow reflects the vectorial process ofribosome formation, the exact structure-function relationships have been a matter ofmuch discussion and are still largely unclear.(For a review, see Scheer and Benavente,1990.)

H ere we describe an approach to localizespecific intermediates for the pre-rRN Amaturation process using in situ hybridi-zation at the ultrastructural level. Thisapproach will allow the correlation of eachpre-rRN A processing step with the definednucleolar substructures. The proceduredescribed can also be applied to thelocalization of other RN A species involvedin ribosome biogenesis, such as 5S rRN A orU3 snRN A (Fischer et al., 1991).

I. Embedding in Lowicryl K4M(according to Carlemalm and Villiger,1989, w ith alterations)

IA. Fixation of material!1 Prepare the following fixation solutions

and keep them on ice:" 4% paraformaldehyde (PFA) in PBS

(137 mM N aCl, 2.7 mM KCl, 7 mMN a2H PO 4, 1.5 mM KH 2PO 4).

" 0.5% glutaraldehyde (GA) plus 4%PFA in PBS.

!2 Take a piece of freshly prepared mouseliver and submerge it in a petri dish filledwith 4% PFA in PBS on ice.

!3 Cut the liver into small cubes with amaximal side length of 1 mm.Note: The smaller the pieces, the betterreagent penetrates them in the proce-dure below.

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II. SectioningThe embedded mouse liver cubes can easilybe recognized in the transparent resinLowicryl K4M. Carlemalm and Villiger(1989) recommend trimming with theultramicrotome and a glass knife to getsmooth surfaces.!1 O btain semithin sections with a glass

knife and ultrathin sections with adiamond knife, according to standardprotocols.Caution: Because of the hydrophilicityof Lowicryl K4M, be careful not to wetthe surface of the block .

!2 Transfer sections to 200 mesh nickel gridswhich have been coated with 0.5%parlodion.

!3 Air dry the sections.Note: N ow the sections are ready forhybridization.

III. Probe preparation!1 Clone restriction fragments from mouse

rDN A.!2 Prepare in vitro transcripts from the

rDN A clones and label them with DIG-UTP according to the protocols inChapter 4 of this manual.

!3 Since probe length might influence theefficiency of in situ hybridization experi-ments, monitor transcript length byelectrophoresis (in formaldehyde-con-taining gels), blotting onto nitrocellulose,and detection with the anti-DIG-alkalinephosphatase conjugate.

!4 If necessary, reduce probe length toapproximately 150 nucleotides by limitedalkaline hydrolysis of the transcriptsaccording to Cox et al. (1984). Briefly, theprocedure is:" Incubate the transcripts with a solution

of 40 mM N aH CO 3 and 60 mMN a2CO 3 at 60°C.

" Calculate the Incubation time asfollows:

L0 – Lft = ________k · L0 · Lf

L0 = initial length of transcript (in kb)Lf = desired probe length (in kb)k = constant = 0.11 kb/min

" Stop the hydrolysis by adding 3 Msodium acetate and acetic acid.

" Precipitate the hydrolyzate with ethanol." Dissolve the precipitate in water.

!1 Prepare Lowicryl K4M as follows (toproduce a medium hard plastic):" Mix 2 g Crosslinker A, 13 g Monomer

B, and 0.075 g Initiator C." Dissolve Initiator C by gentle stirring

or by bubbling dry nitrogen throughthe mixture.

" Cool the mixture to –20°C in thefreezer.

!2 Perform infiltration at about –20°C withsuitably precooled material according tothe following scheme:" 1h with 100% ethanol:K4M (1:1, v/v)." 2 x1 h with K4M." O vernight with K4M." 2 x 3 h with K4M.

ID. EmbeddingGelatin capsules (e.g. Capsulae O perculataeN o. 2) are very suitable as molds forembedding because they are transparent toUV-light and can afterwards easily be cutaway.!1 Cool the gelatin capsules to the

appropriate temperature.!2 Add a few drops of Lowicryl K4M to

each capsule.!3 Drop one object cube into each capsule.

Caution: Do this transfer very carefully.Do not mechanically damage the cube,allow it to dry out, or allow it to warm up.

!4 Fill the capsules with the embeddingmedium.

!5 Let the capsules stand for about half anhour to allow the air bubbles to leave.

IE. Polymerization!1 Before switching on the UV light, turn

the temperature a bit lower to compen-sate for the heat from the light.

!2 Induce polymerization of Lowicryl K4Mby 360 nm long-wave UV radiation(“CS-UV”, Reichert & Jung, CambridgeInstr.) over a five day period. During thepolymerization, do the following:" For about three days, keep the

polymerization temperature constantat any temperature between 0°C and–40°C.

" After three days, allow the temperatureto rise to room temperature with about8 K/h.

Note: For troubleshooting, seeCarlemalm and Villiger (1989).

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Figure 1: In situ hybridization of a digoxigenin-labeled RNA probe complementarytomost of the28 S rRNAregiontoanultrathinsection of mouseliver fixedwith3%formaldehyde and embeddedinLowicryl K4M. The nucleolus as well as the cytoplasm are clearly marked. Label is essentiallyabsent from the mitochondria, demonstrating thespecificity of the method. Cytoplasmic labeling isdue to labeling of the ribosomes, especially theendoplasmatic reticulum-associated ones (Magnification: 25,200x).

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!3 Apply diluted antibody to section andincubate for 1 h.

!4 Wash the sections as follows:" 3 x 5 min with PBST." 6 x 5 min with redistilled water.

!5 Perform silver enhancement by incubatingsectionswith developer and enhancer (fromthe set of silver enhancement reagents) at aratio of 1:1 for 4–20 min (depending on thedesired silver grain size).

!6 Wash the sections 6 x 5 min with redis-tilled water.

!7 Stain with 2% aqueous uranyl acetate(4 min) and Reynolds’ lead citrate for 1 min (Reynolds, 1963) according tostandard procedures.

!8 Evaluate the specimen in the electronmicroscope.

ResultsIn Figure 1, the hybridized probe wasdetected with monoclonal anti-DIG anti-body coupled to 1 nm gold particles. Thesignal was enhanced with silver staining for6 min at room temperature. Silver intensi-fication facilitates visualization of the boundhybridization probe at low magnification.The silver grains scattered throughout thenucleoplasm might indicate transport ofpreribosomal particles from the nucleolus tothe cytoplasm.

In Figure 2, a digoxigenin-labeled riboprobecomplementary to the first 3 kb of the 5' region of the ETS1 was hybridized to aLowicryl section of mouse liver anddetected as described in Figure 1.

IV. HybridizationNote: Perform all the following steps(including the hybridization) in a moistchamber.!1 Prepare hybridization mixture [5 x SSC;

0.1 mg/ml tRN A; DIG-labeled antisensetranscript at a final concentration of10 ng/µl].Note: 1 x SSC contains 0.15 M N aCl,0.015 M sodium citrate.

!2 To monitor the specificity of binding,prepare a “negative” probe by substitut-ing the sense transcript for the antisensetranscript in the hybridization mixtureabove.

!3 Place a droplet of the appropriate hybrid-ization mixture onto Parafilm®.

!4 Incubate the ultrathin sections by puttingthe grid (section-side down) on top of thedroplet.

!5 Perform hybridization for at least 3 h at65°C.

!6 Wash the sections as follows, all at roomtemperature:" 3 x 5 min with 2 x SSC." 2 x10 min with PBST (PBS containing

0.1% Tween® 20).

V. Detection!1 To block nonspecific sites, incubate each

section for 15 min with PBST plus BG(PBST containing 1% bovine serumalbumin, 0.1% CWFS-gelatin).

!2 Dilute 1 nm gold-conjugated anti-DIGantibody 1:30 in PBST plus BG.

Figure 2: Localization of pre-rRNA moleculescontainingthe5' region of the ETS1. For detailsofthe probe, see the text. The survey view showsselective labeling of the fibrillar components of thenucleolus (Magnification: 27,000x).

# #

ReferencesCarlemalm, E.; Villiger, W. (1989) Low temperatureembedding. In: Bullock, G. R.; Petrusz, P. (Eds)Techniques Immunocytochem. 4, 29–44.Cox, K. H.; DeLeon, D. V.; Angerer, L. M.; Angerer, R. C. (1984) Detection of mRNAs in seaurchin embryos by in situ hybridization usingasymmetric RNA probes. Dev. Biol. 101, 485–502.Fischer, D.; Weisenberger, D.; Scheer, U. (1991)Assigning functions to nucleolar structures.Chromosoma 101, 133–140.Reynolds, E. S. (1963) The use of lead citrate athigh pH as an electron-opaque stain in electronmicroscopy. J. Cell Biol. 17, 208–212.Scheer, U.; Benavente, R. (1990) Functional anddynamic aspects of the mammalian nucleolus.BioEssays 12, 14–21.


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T7 RNA polymerase. reactions)Tween® 20 1332 465 5x10 mlBSA Highest quality, lyophilizate 238 031 1 g

238 040 10 gAnti-Digoxigenin- Affinity purified sheep IgG to digoxigenin 1450 590 1 mlGold conjugated with ultra small gold particles

(average diameter ≤ 0.8 nm).Silver Enhancement Detection of colloidal gold particles 1465 350 1 setReagents (anti-DIG-gold) by silver deposition on

the particle surface


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RNA in situhybridization using DIG-labeled cRNA probesH . B. P. M. Dijkman, S. Mentzel, A . S. de Jong, and K. J. M. AssmannDepartment of Pathology, N ijmegen University H ospital, N ijmegen, The N etherlands.

I. Probe selectionChoosing the type of probe (i.e., DN A,RN A, or oligonucleotide) is essential for a good final result. For the optimization ofthe probe labeling, we have tested fourmethods of incorporating the DIG label:

! Direct labeling of cDN A molecules usingthe DIG DN A Labeling Kit*.

! DIG labeling of oligonucleotides usingthe DIG O ligonucleotide Tailing Kit*.

! DIG labeling of PCR products accordingto the method of H annon et al. (1993).

! DIG labeling of cRN A molecules usingthe DIG RN A Labeling Kit*.

The RN A labeling method gave by far thebest results. Therefore, we provide somehints on how to transcribe DIG-labeledcRN A molecules:

! For cRN A probe synthesis, subclone acDN A molecule in an appropriate vector.The vector should have sequencesflanking the insert that allow the insert tobe transcribed. Typically, commerciallyavailable vectors have SP6 and T7 RN Apolymerase sites flanking the multiplecloning sites.

! Regulate the length of the cDN Amolecule since this greatly affects thehybridization efficiency. Usually, a cDN Alength between 200 and 500 nucleotidesgives the best results, allowing efficienthybridization and good penetration of thetissue.

! When a molecule of interest is expressed asa high-copy RN A but the target RN A ismasked by proteins, make probes between100 and 200 nucleotides long for betterpenetration (Figure 1).

In recent years, the in situ hybridization(ISH ) technique has found widespreadapplication in both basic science and diag-nostic clinical research. The ISH techniquehas frequently been used to localize specif-ic genes on metaphase chromosomes andto detect viral and bacterial genomes ininfected tissues. The RN A in situ hybridi-zation (RISH ) technique for the examina-tion of mRN A expression has gained lessattention due to the many technical problemsassociated with this technique.

In this report, we present a step-by-stepprotocol for a nonradioactive RISH tech-nique on frozen sections using digoxigenin-labeled copy RN A (cRN A) probes. Wedemonstrate this technique on frozensections of mouse kidney using DIG-labeledcRN A probes for the ectoenzyme amino-peptidase A, a low-copy RN A (Assmann etal., 1992). For a high-copy RN A, we studiedhuman psoriatic epidermis using a DIG-labeled cRN A probe for elafin/SKALP, aninhibitor of leukocyte elastase and proteinase3 (Alkemade et al., 1994). This protocol hasbeen optimized to give strong hybridizationsignals, even with low-copy mRN Amolecules.

For a full discussion of each step of thisprotocol, along with more hints on enhanc-ing the result of this often laborious andtroublesome technique, see the previouslypublished version of this report (Dijkman etal., 1995a; 1995b).


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II. Probe labeling"1 After subcloning the cDN A molecule,

linearize the circular vector with re-striction enzymes that cut the multiplecloning site in 2 orientations, allowingsense and antisense synthesis.Caution: Control this linearization stepcarefully by monitoring the digestionwith agarose gel electrophoresis. Circularmolecules that are left in the digestionmixture affect the transcription efficiency.

"2 Extract the linearized molecules from thedigestion mixture by phenol extraction.

"3 Label the transcripts from the linearizedmolecules with DIG-labeled nucleotidesand the DIG RN A Labeling Kit*according to the instructions given in thekit, but with the following modifications:! Perform the SP6 polymerase incuba-

tion at 40°C instead of 37°C.! Precipitate the labeled molecules over-

night at –20°C, rather than 30 min at–70°C.

! Monitor the transcription reaction byagarose gel electrophoresis to check forcorrect cRN A probe length.

"4 Monitor probe labeling by spottingdiluted aliquots of the labeled cRN Aprobes on nylon membranes and ana-lyzing with the DIG Luminescent Detec-tion Kit*. See also Chapter 4, page 51.Note: The sense and antisense cRN Aprobes should be labeled equally asefficiently as the control DN A includedin the DIG RN A Labeling Kit*.

"5 If necessary, adjust the concentration ofthe labeled sense and antisense cRN Aprobes so they contain equal amounts oflabel.

"6 Aliquot the cRN A probes in poly-propylene tubes at –70°C. Note: Repeated freez ing and thawingaffects the labeled cRN A probe.

Figure 1: RNA in situ hybridization on a frozensection of human psoriatic epidermis with DIG-labeled cRNA probes coding for human SKALP. Panel a: An antisense cRNA probe (150 bp) wasused on a 10 µm thick section (magnificationx125).Panel b: An antisense cRNA probe (150 bp) wasused on a 20 µm thick section (magnificationx500).Panel c: A sense cRNA probe (150 bp) was used ona 10 µm thick section (magnificationx270). Intensestaining of both the stratum spinosum and stratumgranulosum is observed with the antisense probe.

a

b

c


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I NDEXCONTENTS

Figure 2: RNA in situ hybridization on a frozenkidneysectionwitha DIG-labeledcRNAprobeformouseaminopeptidaseA(without delipidization).The section was 10 µM thick and was taken from a male BALB/c mouse. Note the many nonspecificlipid vesicles in the section. The specific hybridi-zation signal appears in cells of the glomerulus (arrows). Magnification, 600 x.

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"5 (O ptional) To minimize nonspecificbackground caused by lipid vesicles, dothe following (all at room temperature):! Delipidize the sections by extracting

them for 5 min in chloroform.! Dry the section to evaporate the

chloroform.Note: This delipidation step may beomitted in pilot studies on a new tissue.

"6 Fix the tissue sections as follows (all atroom temperature):! Incubate tissue in PBS containing 4%

paraformaldehyde for 7 min.! Wash 1 x 3 min with PBS.! Wash 2 x 5 min with 2 x SSC.

Note:1 x SSC contains 150 mM N aCl,15 mM sodium citrate; pH 7.2.

III. Preparation of tissue sectionsNote: For detection of low-copy RN Amolecules, always prepare frozen sections,since paraffin embedding causes a loss ofapproximately 30% of the RN A. Themethod described here has been optimizedfor frozen sections."1 For the processing of tissue, clean all

knives and other materials with RN aseZAP (AMBIO N ) and work as asepticallyas possible.

"2 Remove the tissue from the animal,immediately snap-freeze the tissue, andstore it in liquid nitrogen. Work quicklyto avoid degradation of RN A (Barton etal., 1993).

"3 Cut 10 µm thick frozen sections.Note: To get a higher signal, cut sectionsthicker than 10 µm. For better signallocalization, cut sections thinner than10 µm.

"4 Mount tissue directly on Superfrost Plusslides (Menzel Gläser, O mnilabo, Breda,The N etherlands) to prevent detachmentof the section during the RISHprocedure.

IV. Pretreatment of slidesCaution: Prepare all solutions forProcedures IV w ith water that has beentreated w ith 0.1% DEPC."1 Directly after mounting, heat the sections

on a stove for 2 min at 50°C to fix theRN A in the tissue.Note:Vary the time (from 10–120 s) andtemperature (from 50°–90°C) of thefixation step to accommodate differenttypes of tissue and RN A.

"2 Dry the sections for 30 min."3 Circle the sections with a silicone pen

(DAKO A/S, Glostrup, Denmark) toprevent smudging of the substrate.

"4 Use the following criteria to decide thenext step of the procedure:! I f the target tissue has lipid vesicles

(Figure 2) that interfere with the RISHdetection, go to Step 5.

! I f the target tissue does not have lipidvesicles that interfere with the RISHdetection, go to Step 6.

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5

VI. Immunological detection"1 Wash the sections for 5 min at room

temperature with 100 mM Tris-H Cl (pH7.5), 150 mM N aCl.

"2 Incubate the sections for 30 min at roomtemperature with blocking buffer[100 mM Tris-H Cl (pH 7.5), 150 mMN aCl; saturated with blocking reagent].

"3 Prepare a 1:200 dilution of alkaline phos-phatase-conjugated anti-DIG antibody(polyclonal, Fab fragments, from sheep)in blocking buffer (from Step 2).

"4 Incubate the sections for 120 min at roomtemperature with the diluted anti-DIGantibody conjugate prepared in Step 3.

"5 Wash the sections as follows:! 2 x 5 min at room temperature with

100 mM Tris-H Cl (pH 7.5), 150 mMN aCl.

! 1x10 min at room temperature with100 mM detection buffer [Tris-H Cl(pH 9.5), 100 mM N aCl, 50 mMMgCl2].

"6 Cover the sections with detection buffercontaining 0.18 mg/ml BCIP, 0.34 mg/mlN BT, and 240 µg/ml levamisole. Incubatefor 16 h at room temperature (fordetection of low abundance RN A).Note: This development should becarried out w ith the slides standing upin a small container to prevent non-specifically converted substrate fromfalling onto the section.

"7 Stop the color reaction by washing thesections for 5 min in 10 mM Tris (pH 8),1 mM EDTA.

VII. Counterstain"1 Wash the sections for 5 min with distilled

H 2O at room temperature."2 Counterstain the sections for 5–10 min

with 1% methylene green at 37°C."3 Repeat wash (from Step 1)."4 Mount coverslips in Kaiser’s solution

(available from Merck).

V. Prehybridization, hybridization, and posthybridizationCaution: Prepare all solutions forProcedures V w ith water that has beentreated w ith 0.1% DEPC."1 Prehybridize each section for 60 min at

37°C in 100 µl hybridization buffer [4 x SSC; 10% dextran sulfate; 1 xDenhardt’s solution (0.02% Ficoll® 400,0.02% polyvinyl pyrolidone, 0.02%bovine serum albumin); 2 mM EDTA;50% deionized formamide; 500 µg/mlherring sperm DN A].Note: You may vary the temperature ofthe prehybridization and hybridizationsteps between 37°C and 50°C .

"2 Perform hybridization as follows:! Remove and discard the buffer from

the prehybridization step.! Cover each section with 100 µl hybridi-

zation buffer containing 200 ng/ml ofDIG-labeled cRN A probe.Caution: Do not use coverslips, sincethey decrease the signal up to fourfold.Caution: A probe concentration of200 ng/ml holds only if the cRN A waslabeled efficiently. I f, in Procedure I I ,the cRN A probe was not labeled asefficiently as the controls from theDIG RN A Labeling Kit*, adjust theconcentration of the cRN A probe inthe hybridization mixture.

! Incubate for 16 h at 37°C.Note: You may vary the temperatureof the prehybridization and hybridi-zation steps between 37°C and 50°C.

"3 After the hybridization, wash unboundcRN A probe from the section as follows:! 1 x 5 min with 2 x SSC at 37°C.

Note: To make the wash morestringent and wash away nonspecif-ically bound cRN A probe, lower thesalt concentration or increase theformamide concentration in thewashing buffer and perform thewashing step at a temperature 5°Cbeneath the melting temperature ofthe probe.

! 3 x 5 min with 60% formamide in 0.2 x SSC at 37°C.

! 2 x 5 min with 2 x SSC at room tem-perature.


I NDEXCONTENTS

Results and discussionFigure 3 shows a typical RISH signalobtained with this protocol and a labeled antisense cRN A probe coding for mouseaminopeptidase A. The protocol includedthe optional delipidation step with chloro-form (Procedure IV, Step 5). Figure 4 showsthe control reaction with the labeled sensecRN A probe.

We have provided a standard protocol forRISH on low- and high-copy RN Amolecules. Using this protocol, one shouldbe able to perform RISH on each RN Amolecule of interest, with only minormodifications depending on the abundanceof the RN A of interest.

Figure 4: RNA in situ hybridization on a frozenkidney section from the same mouse used inFigure 3 with a DIG-labeled sense cRNA probefor mouse aminopeptidase A. As in Figure 3, the section was 10 µm thick and was taken from amale BALB/c mouse. The section was pretreatedwith chloroform prior to the hybridization. No signalcould be seen, indicating the specificity of theantisense hybridization signal seen in Figure 3.(magnification, 600x).

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AcknowledgmentsThe cDN A clone coding for the mouse ami-nopeptidase A cDN A sequence was kindlyprovided by Dr. Max D. Cooper from theUniversity of Alabama at Birmingham, USA(Wu et al., 1990). The cRN A probe forelafin/SKALP was kindly provided by Dr. J.Schalkwijk, department of Dermatology,University H ospital N ijmegen, TheN etherlands.

Figure 3: RNA in situ hybridization on a frozenkidney section with a DIG-labeled antisensecRNA probe for mouse aminopeptidase A (afterdelipidization). The section was 10 µm thick and was taken from a male BALB/c mouse.Pretreatment of the section with chloroform prior tothe hybridization delipidized the section andreduced a nonspecific signal previously observed in lipidvesicles (Figure 2). The specific hybridization signal(arrows) was undiminished by the chloroform

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5

ReferencesAlkemade, J. A. C.; Molhuizen, H. O. F.; Ponec, M.;Kempenaar, J. A.; Zeeuwen, P. L. J. M.; de Jongh, G. J.; van Vlijmen-Willems, I. M. J. J.; van Erp, P. E. J.;van de Kerkhof, P. C. M.; Schalkwijk, J. (1994)SKALP/elafin is an inducible proteinase inhibitor inhuman epidermal keratinocytes. J. Cell Sci. 107, 2335–2342.Assmann, K. J. M.; van Son, J. P. H. F.; Dijkman, H. B. P. M.; Koene, R. A. P. (1992) A nephritogenicrat monoclonal antibody to mouse aminopeptidaseA. Induction of massive albuminuria after a singleintravenous injection. J. Exp. Med. 175, 623–636.Dijkman, H. B. P. M.; Mentzel, S.; de Jong, A. S.;Assmann; K. J. M. (1995a) RNA In Situ hybridizationusing digoxigenin-labeled cRNA probes. Biochemica(U.S. version) No. 2 [1995], 23–27.

Dijkman, H. B. P. M.; Mentzel, S.; de Jong, A. S.; Assmann; K. J. M. (1995b) RNA In Situ hybridizationusing digoxigenin-labeled cRNA probes. Biochemica(worldwide version) No. 2 [1995], 21–25.Barton, A. J. L.; Pearson, R. C. A.; Najlerahim, A.;Harrison, P. J. (1993) Pre- and postmorteminfluences on brain RNA. J. Neurochem. 61, 1–11.Hannon, K.; Johnstone, E.; Craft, L. S.; Little, S. P.;Smith, C. K.; Heiman, M. L.; Santerre, R. F. (1993)Synthesis of PCR-derived, single-stranded DNAprobes suitable for in situ hybridization. Anal. Biochem. 212, 421–427.Wu, Q.; Lahti, J. M.; Air, G. M.; Burrows, P. D.;Cooper, M. D. (1990)Molecular cloningof themurineBP-1/6C3 antigen: a member of the zinc-dependentmetallopeptidase family. Proc. Natl. Acad. Sci. USA87, 993–997.



T7 RNA polymerases. reactions)EDTA Powder 808 261 250 g

808 270 500 g808 288 1 kg

DNA, from herring lyophilized sodium salt 223 646 1 gspermBlocking Reagent For nucleic acid hybridization 1096 176 50 gAnti-Digoxigenin-AP Anti-Digoxigenin, Fab fragments 1093 274 150 U

conjugated with alkaline phosphatase (200 µl)Tris For preparation of buffer solutions 127 434 100 g

708 968 500 g708 976 1 kg

NBT solution 100 mg/ml nitroblue tetrazolium salt in 1383 213 3 ml (300 mg)70% (v/v) dimethylformamide (dilute prior

to use)BCIP solution 50 mg/ml 5-bromo-4-chloro-3-indolyl 1383 221 3 ml (150 mg)

phosphate (BCIP), toluidinium salt in100% dimethylformamide


I NDEXCONTENTS

Localization of the expression of the segmentationgene hunchback in Drosophila embryos with DIG-labeled DNA probesDr. D . Tautz , Institute for Genetics and Microbiology, University of Munich, Germany.

158

Procedures for In Situ H ybridization to Chromosomes, Cells, and Tissue SectionsWhole mount ISH

5

I. Preparation of embryosNote: All embryos in this study came fromwild-type Drosophila flies and were col-lected on apple juice agar plates atintervals of 0–4 h at 25°C (Wieschaus andN üsslein-Volhard, 1986)!1 Collect the embryos in small baskets.!2 Wash embryos with water.!3 Dechorionate embryos for about 2–3 min

in a solution of 50% commercial bleach(about 5% sodium hypochlorite) in water.

!4 Wash the embryos with 0.1% Triton® X-100.

!5 Fix the embryos either with formald-ehyde (Procedure IIA) or paraformald-ehyde (Procedure IIB).

IIA. Formaldehyde fixation!1 Transfer each embryo into a glass

scintillation vial containing 4 ml offixation buffer [0.1 M H epes, pH 6.9;2 mM magnesium sulfate, 1 mM EGTA(from a 0.5 M stock EGTA solution thathas been adjusted to pH 8 with N aO H )].

!2 Add to the scintillation vial 0.5 ml 37%formaldehyde solution and 5 ml heptane.

!3 Shake the vial vigorously for 15–20 minto maintain an effective emulsion of theorganic and the water phase.

!4 Remove the lower phase and add 10 mlmethanol." I f the embryos sink to the bottom at

this stage, proceed to Step 5." If the embryos do not sink to the

bottom at this stage, remove the upperphase (heptane) and add more methanol.

!5 Store the fixed embryos in methanol (upto several weeks) in the refrigerator.

In situ hybridization to RN A on sectionsof early embryos provided one of the mostsignificant advances for the analysis ofspatially regulated transcripts of segmen-tation genes in Drosophila. It was thereforesoon adapted to other systems. Theprocedure is, however, rather tedious andtime-consuming. It depends on the qualityof the sections and particularly on thequality of the probe.

In contrast, antibody staining of the proteinproducts from these genes in whole embryossimplified the procedure and allowed a muchhigher resolution. H owever, certain regula-tory events have to be analyzed at the RN Alevel. Also antibody production itself issomewhat time consuming. We have thereforesought to develop an in situ hybridizationtechnique which offers the same advantages asantibody staining. We found the use ofdigoxigenin-labeled probes to be highlysuccessful for this purpose. These probesallow a resolution which is directly compar-able to antibody staining in embryos, andboth the sensitivity and the speed is superiorto conventional radioactive methods.

The method is not limited to applications inDrosophila, but is apparently suitable for allkinds of tissues, at least for those in whichantibody staining has been successful. Wehave already developed a protocol for thestaining of specific cells in whole H ydraanimals, which required only minormodifications (E. Kurz and C. David,University of Munich, Dept. of Zoology).

The procedure given below has been pub-lished (Tautz and Pfeiffle, 1989).

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III. PretreatmentCaution: In the steps below, use thenormal precautions for avoiding potentialRN ase contamination.!1 Treat the PBS for the following steps with

diethylpyrocarbonate (20 µl per 500 mlPBS), then autoclave the treated PBS.

!2 Place each fixed embryo in a 1.5 mlmicrocentrifuge tube. Perform the incu-bations in Steps 3–8 below at roomtemperature on a revolving wheel.

!3 Wash each embryo 3 x 5 min in 1 ml PBT(PBS plus 0.1% Tween® 20).Note: Tween reduces non-specific adhesionof the embryos to the plastic surfaces.

!4 Incubate the embryo for 3–5 min in 1 ml ofPBS containing 50 µg/ml Proteinase K.

!5 Stop the digestion by removing theProteinase K solution, adding 1 ml PBTcontaining 2 mg/ml glycine to theembryo. Incubate for 2 min.Caution: The proteinase digestion timeis critical. I f it is too short, the back -ground increases and sensitiv ity is lost.I f it is too long, the embryos burstduring the subsequent steps.

!6 After the Proteinase K digestion, washthe embryo 2 x 5 min in 1 ml PBT.

!7 Refix for 20 min in 1 ml PP (4%paraformaldehyde in PBS).

!8 Wash 3 x 10 min in 1 ml PBT.

IV. Probe labelingThe probes are DN A restriction fragmentsisolated from agarose gels. Label themaccording to the random priming methoddescribed in Chapter 4 of this manual.

IIB. Paraformaldehyde fixationNote: This protocol is more laborious, butleads, in some cases, to a lower backgroundand to better preservation of the morphology.!1 Transfer the embryos into a glass

scintillation vial containing 1.6 ml offixation buffer [100 mM H epes, pH 6.9;2 mM magnesium sulfate, 1 mM EGTA(from a 500 mM stock EGTA solutionthat has been adjusted to pH 8 withN aO H )].

!2 Liquify a 20% stock solution of para-formaldehyde in water as follows:" Remove the stock 20% paraformalde-

hyde from its storage at –20°C." H eat the stock to 65°C." N eutralize with a little N aO H .

!3 From the liquified stock 20% solution ofparaformaldehyde, remove 0.4 ml andadd it to the scintillation vial.

!4 Also add 8 ml heptane to the vial.!5 Shake the vial and treat with methanol as

in Steps 3 and 4 in Procedure IIA above.!6 Transfer each embryo to a 1.5 ml micro-

centrifuge tube containing ME [90%methanol, 10% 0.5 M EGTA] .

!7 Prepare PP solution (4% paraformalde-hyde in PBS).Note: PBS contains 130 mM N aCl and10 mM sodium phosphate; pH 7.2.

!8 Refix the embryos and dehydrate bypassage through the following series ofsolutions containing ME and PP:" 5 min in ME:PP (7:3)." 5 min in ME:PP (1:1)." 5 min in ME:PP (3:7)." 20 min in PP alone.

!9 Wash the embryos in PBS for 10 min anddo either of the following:" Use the fixed embryos in Proce-

dure III.or

" If the fixed embryos are not to be usedimmediately, dehydrate them in aseries of ethanol solutions (30% , 50% ,then 70% ethanol) and store them at–20°C.Caution:Dehydrated, stored embryosmust be rehydrated before they canbe used in Procedure I I I .

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VI. Detection!1 Prepare a fresh dilution (1:2000 to

1:5000) of alkaline phosphatase-conjugated anti-DIG-antibody in PBT.Note: The antibody may be preab-sorbed for 1 h w ith fixed embryos toreduce the background.

!2 Incubate each embryo with 500 µl freshlydiluted antibody conjugate for 1 h atroom temperature on a revolving wheel.

!3 Wash the embryo as follows:" 4x20 min in PBT." 3x5 min in color buffer [100 mM

N aCl, 50 mM MgCl2, 100 mM Tris(pH 9.5), 1 mM levamisole, 0.1%Tween® 20].Note: Levamisole is a potentinhibitor of lysosomal phosphatases.

!4 Bring the embryos into a small dish with1 ml of color buffer. Add to this 4.5 µl N BT and 3.5 µl BCIP with thor-ough mixing.

!4 Develop the color for 10–60 min in thedark.Note: Color development mayoccasionally be controlled under thebinocular microscope and stopped inPBT before background develops.

!6 Dehydrate the embryos in a series ofethanol solutions and mount in GMM(Lawrence et al., 1986).

ResultsFigure 1 shows typical results obtained withthe above protocol. Panel a shows threeembryos at different developmental stageswhich represent the dynamic expressionpattern of hunchback. The embryo on thetop right in Panel a shows the maternalgradient of hunchback RN A distribution;the embryo at the left the primary zygoticexpression in two domains; and the middleembryo, the late zygotic expression.

In Panel b, an enlarged section of an embryoshows the predominant cytoplasmic loca-tion of the hybridization signal. The sectionof the embryo corresponds to the framedepicted in Panel a although the photo wastaken of a different embryo.

Panel c shows an embryo after gastrulation,where individual cells of the developingnervous system express hunchback.

V. Hybridization!1 Prepare the hybridization solution (H S)

[50% formamide; 5 x SSC (where 1 x SSCcontains 150 mM N aCl, 15 mM sodiumcitrate); 50 µg/ml heparin, 0.1% Tween®

20; and 100 µg/ml sonicated and dena-tured salmon sperm DN A].Note: H S may be stored at –20°C.

!2 Wash the embryos as follows:" 20 min in 1:1 H S:PBT." 20–60 min in H S.

!3 Prehybridize for 20–60 min in H S at45°C in a water bath.

!4 H eat denature the probe in the presenceof 10 µg sonicated salmon sperm DN A.Note: The salmon sperm DN A shouldeffectively compete out simple sequencesand thus reduce the background.

!5 Dilute the probe to approximately 0.5 µglabeled probe per ml in H S.

!6 Remove most of the prehybridizationsupernatant from the embryo, then addthe heat-denatured probe and thoroughlymix.

!7 H ybridize overnight at 45°C in a waterbath without shaking.

!8 Wash the embryos at room temperaturein each of the following solutions:" 20 min in H S." 20 min in 4:1 H S:PBT." 20 min in 3:2 H S:PBT." 20 min in 2:3 H S:PBT." 20 min in 1:4 H S:PBT." 2 x 20 min in PBT.Note: Use a less extensive washingprotocol if probe produces a low back -ground.

I NDEXCONTENTS

Figure 1: In situ hybridization onwhole Drosophila embryos usinga probe for the segmentation genehunchback. Optical sections wereobtained by appropriate focusing.For details of the panels, see text.Panel a (The bar at the top represents approx. 100 µm.)Panel b (The bar at the top represents approx. 5 µm.)

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ReferencesLawrence, P. A.; Johnston, P.; Morata, G. (1986)Methods of marking cells. In: Roberts, D. B. (ed.)Drosophila, A Practical Approach. Oxford: IRL Press,pp. 229–242.Tautz, D.; Pfeiffle, C. (1989) A nonradioactive in situhybridization method for the localization of specificRNAs in Drosophila embryos reveals a translationalcontrol of the segmentation gene hunchback. Chromosoma (Berl.) 98, 81–85.

a c

b

nuclei

cytoplasm

yolk

Reagent Description Cat. No. Pack size Triton® X-100 Vicous, liquid 789 704 100 mlHepes Purity: 98% (from N) 223 778 100 g

737 151 500 g242 608 1 kg

EGTA Purity: > 98% 1093 053 50 gTween® 20 1332 465 5x10 mlProteinase K Lyophilizate 161 519 25 mg

745 723 100 mg1000 144 500 mg1092 766 1 g

DIG-High Prime*, **, *** Premixed solution for 40 random-primed 1585 606 160 µl DNA labeling reactions with DIG-11-dUTP, (40 labelingalkali-labile reactions)

Anti-Digoxigenin-AP 750 units/ml Anti-Digoxigenin, Fab 1093 274 150 U fragments conjugated to alkaline (200 µl)phosphatase




Wieschaus, E.; Nüsslein-Vollhard, C. (1986) Looking at embryos. In: Roberts, D. B. (ed.) Drosophila, A Practical Approach. Oxford: IRLPress, pp. 199–228.


#

***EP Patent 0 324 474 granted toand EP Patent application 0 371 262 pending for BoehringerMannheim GmbH.

***This product or the use of thisproduct may be covered by one or more patents of BoehringerMannheim GmbH, including thefollowing: EP patent 0 649 909(application pending).


I NDEXCONTENTS

Detection of even-skipped transcripts in Drosophilaembryos with PCR/DIG-labeled DNA probesDr. N . Patel and Dr. C . Goodman, Carnegie Institute of Washington, Embryology Department, Baltimore, Maryland, USA.

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I. Probe labeling!1 Prepare the following stock solutions:

" 10 x concentrated reaction mix:500mMKCl; 100 mM Tris-H Cl, pH 8.3;15 mM MgCl2; 0.01% (w/v) gelatin.

" 5 x concentrated dN TP mix: 1 mMdATP, 1 mM dCTP, 1 mM dGTP,0.65 mM dTTP, 0.35 mM digoxigenin-11-dUTP.

" A primer stock solution containingeither 30 ng/µl (approx. 5.3 mmol)primer 1 (e.g. generated by SP6 RN Apolymerase) or 30 ng/µl (approx. 5.3 mmol) primer 2 (e.g. generated byT7 RN A polymerase).

!2 With a restriction enzyme, linearize “dualpromotor vector DN A” containing theinsert, as one would to make run-offRN A transcripts.Note: The two different primers can beused to create an antisense strand and asense strand (as control).

!3 H eat inactivates the restriction enzyme.!4 Dilute the linearized DN A with water to

a final concentration of about 100–200 ng/µl.Note: I f the insert is much over 3 kb,the probe produced w ill probably notrepresent the entire insert.

!5 Set up the following reaction mix:" 9.25 µl water." 2.5 µl 10 x concentrated reaction

mixture." 5.0 µl 5 x concentrated dN TP mix." 5.0 µl primer 1 or 2 (from 30 ng/µl

stock)." 2.0 µl linearized DN A (100–200 ng/µl).

!6 Add 40 µl mineral oil, centrifuge, thenboil the mix for 5 min.

!7 To the mix, add 1.25 µl of 1 unit/µl TaqDN A Polymerase (1.25 units of TaqPolymerase). [Final volume of reactionmix with Taq is 25 ml.]

!8 Mix the contents of the reaction tube andthen centrifuge for 2 min.

This protocol has been used to detect thetranscript distribution of a number of genesby in situ hybridization, including even-skipped and seven-up, in whole mountDrosophila embryos, and engrailedAntennapedia in whole mount grasshopperembryos. The in situ hybridization anddetection was performed essentiallyaccording to the protocol of Tautz andPfeiffle (1989).

This laboratory uses PCR rather thanrandom primed labeling to prepare DIG-labeled probes because:" A much larger quantity of probe can be

made with the same amount of startingnucleotide.

" The ratio of labeled DN A to unlabeledstarting material is much higher(especially important when transcripts tobe detected are not very abundant).

" PCR can produce strand-specific copies.

Disadvantage:

" It is more difficult to control the probesize.

This laboratory has also found that biotin-16-dUTP incorporated in the same way anddetected with streptavidin-alkaline phos-phatase is about three- to fivefold lesssensitive than DIG-labeled probes in in situhybridization experiments.

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II. Evaluation of labeling reaction!1 Prepare an aliquot of the probe as follows:

" Remove 1 µl of probe from thereaction mix.

" Add 5 µl of 5 x SSC." Boil 5 min." Q uick cool on ice." Centrifuge.

!2 Spot 1–2 µl of the probe aliquot onto asmall nitrocellulose strip cut to fit into a1.5 ml microcentrifuge tube or a 5 mlsnap cap tube.

!3 Bake the filter between two sheets offilter paper in an 80°C vacuum oven for30 min.Caution: The residual formamide maycause the nitrocellulose to warp. I f thisis a problem, reduce the time in thebak ing oven or do this spot test beforethe second precipitation (Procedure I ,Step 12).

Note: Unincorporated nucleotide bindsonly slightly to the nitrocellulose.

!4 Treat the baked filter as follows:" Wet the filter with 2 x SSC." Wash filter 2 x 5 min in PBT (1 x PBS,

0.2% BSA, 0.1% Triton® X-100)." Place filter into a 1.5 ml micro-

centrifuge tube or 5 ml snap cap tube.!5 Detect the labeled probe as follows:

" Block the filter by incubating it 30 min in PBT.

" Incubate in PBT for 30–60 min with a1:2000 dilution (in PBT) of alkalinephosphatase-conjugated anti-DIG anti-body.

" Wash 4 x 15 min in PBT." Wash 2 x 5 min in a solution contain-

ing 100 mM N aCl; 50 mM MgCl2;100 mM Tris, pH 9.5; 0.1% Tween® 20.

Note: Levamisole is not needed.!6 Develop color with N BT and BCIP as

described in the appropriate BoehringerMannheim pack insert.

!7 Stop the reaction when the spots arevisible.Note: Spots should be v isible w ithin afew minutes and dark after 10–15 min.

!9 Incubate for 30 cycles in the PCR thermalcycler under the following conditions: " Denaturation at 95°C for 45 s." Annealing at 50°–55°C for 30 s.

Note: Annealing temperature dependson length of primer. Use 55°C for a21-mer.

" Elongation at 72°C for 1.0–1.5 min.Note: Elongation time depends onlength of insert. Use 1 min for 1.0 kbor less, 1.5 min for 2.5 kb or more.

!10 After the PCR run, add 75 µl distilled H 2Oto the reaction tube, then centrifuge it.

!11 Remove 90–95 µl of the reaction mixfrom beneath the oil.

!12 To recover the DN A, do an ethanolprecipitation as follows:" Add N aCl to a final concentration of

0.1 M." Add 10 µg of glycogen or tRN A as a

carrier (0.5 µl of a 20 mg/ml stock)." Add 3 volumes of 100% EtO H ." Mix well and leave at –70°C for

30 min." Centrifuge." Wash pellet with 70% ethanol." Dry under vacuum.O ptional: Perform a second ethanolprecipitation as above.

!13 Resuspend DN A pellet in 300 µl ofhybridization buffer [50% formamide;5 x SSC (where 1 x SSC contains 150 mMN aCl, 15 mM sodium citrate); 50 µg/mlheparin, 0.1% Tween® 20; and 100 µg/ml sonicated and denatured salmonsperm DN A] (according to Tautz andPfeifle, 1989).

!14 To reduce the size of the single-strandedDN A, boil the probe for 40–60 min. Note: For efficient penetration of andhybridization to the embryos, theaverage probe length should be about50–200 bp.

!15 Dilute the probe as much as tenfoldbefore use.Note: O ptimal dilution varies depend-ing on the abundance of the transcriptand background staining. We recom-mend using the probe either undiluted(original 300 µl) or diluted up to three-fold for the initial experiment.

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Figure 1: Detection of the even-skippedphenotype on blastoderm stage Drosophilaembryos. Panel a shows an in situ hybridizationusing the Drosophila even-skipped gene as probe. Panel b demonstrates the even-skipped productusing the specific antibody as probe. The sevenstripe pattern is associated with the even-skippedphenotype.

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ReferenceTautz, D.; Pfeiffle, C. (1989) A nonradioactive in situhybridization method for the localization of specificRNAs in Drosophila embryos reveals a translationalcontrol of the segmentation gene hunchback. Chromosoma (Berl.) 98, 81–85.


III. Preparation of embryos,hybridization and detectionPerform the preparation of the embryos,subsequent hybridization of the DIG-PCRprobe, and immunological detection asdescribed in the Tautz article, “Localizationof the expression of the segmentation genehunchback in Drosophila embryos withdigoxigenin-labeled DN A probes,” page158 in this manual.

Results

a b

Reagent Description Cat. No. Pack size PCR DIG Probe For generating highly sensitive probes 1636 090 1 KitSynthesis Kit* labeled with DIG-dUTP (alkali-labile) in (25 reactions)

the polymerase chain reaction (PCR)using a ratio of 1+2 (DIG-dUTP:dTTP)

Tween® 20 1332 465 5x10 mlGlycogen From rabbit liver 106 089 500 mgBSA Highest quality, lyophilizate 238 031 1 g

238 040 10 gTriton® X-100 Vicous, liquid 789 704 100 mlAnti-Digoxigenin-AP 750 units/ml Anti-Digoxigenin, Fab 1093 274 150 U

fragments conjugated to alkaline (200 µl)phosphatase




#

*This product is sold under licensingarrangements with RocheMolecular Systems and The Perkin-Elmer Corporation. Purchaseof thisproduct is accompanied by alicense to use it in the PolymeraseChain Reaction (PCR) process inconjunction with an AuthorizedThermal Cycler. For completelicense disclaimer, see inside back

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Wholemount fluorescenceinsituhybridization(FISH)of repetitive DNA sequences on interphase nuclei ofthe small cruciferous plant Arabidopsis thalianaSerge Bauwens1 and Patrick Van O ostveldt2

1 Laboratory for Genetics, Gent University, Gent, Belgium2 Laboratory for Biochemistry and Molecular Cytology, Gent University, Gent, Belgium

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!3 Pipette onto germination medium (1xMurashige and Skoog salt mixture (FlowLaboratories, USA); 0.5 g/L 2-(N -morpholino)ethane sulphonic acid(MES), pH 5.7 (adjusted with 1 MKO H ); 0.8% (w/v) Bacto-agar (DifcoLaboratories, USA).

!4 Allow the seeds to germinate at roomconditions for 4 days.

!5 Transfer part of the seedlings to soil.!6 Grow plants under continuous light

conditions at desk temperature.!7 H arvest flowers and inflorescences after

3–4 weeks.

II. Tissue fixation!1 Place approximately 30–40 seedlings or a

few flowers or inflorescences in a glassvial containing:4.365 ml fixation buffer [1.1 x PBS; 0.067 M EGTA, pH 7.5 (adjusted withN aO H )].Note: 10 x PBS contains 1.3 M N aCl,0.0027 M KCl, 0.07 M N a2H PO 4, 0.03 MN aH 2PO 4; pH 7.2." 0.135 ml 37% formaldehyde (Sigma,

USA)." 0.5 ml DMSO .Note: Final concentrations in v ial are1% formaldehyde and 10% DMSO .

!2 Rock the glass vial for 25 min at roomtemperature.

!3 Remove the fixative and rinse as follows:" 2 x with 5 ml methanol." 4 x with 5 ml ethanol.

!4 Discard the last ethanol wash, then coversample with a final 5 ml ethanol.

!5 Leave sample at –20°C for 2–4 days.Caution: Keeping the material forlonger periods of time in ethanol makesit brittle.

H ybridizing fluorescently labeled DN Aprobes in situ to the chromatin of interphasenuclei in whole mounts allows the study ofnuclear architecture in morphologically wellpreserved specimens. Fluorescent labeling ofthe DN A probes (either directly orindirectly) allows the simultaneous, butdifferential, detection of several sequences(e.g. Lengauer et al., 1993; N ederlof et al.,1990). Also, fluorescent signals can beimaged by confocal microscopy so thatstacks of optical section images can berecorded through the whole mounts.Multiple labeling FISH on whole mountstherefore allows the study of the relativepositions of different chromosomes orchromosome segments in individualinterphase nuclei as well as between nuclei ofrelated cells.

In the experiments presented here, twotandemly repeated sequences, rDN A and a500 bp repeat sequence, were hybridized tointerphase nuclei of seedlings and flowers(inflorescences) of the small cruciferousplant Arabidopsis thaliana. The procedureused in the experiments, based on theprotocols published by Ludevid et al. (1992)and Tautz and Pfeifle (1989), was describedearlier by Bauwens et al. (1994).

I. Seed sterilization and germinationNote: Procedure I is based on the protocolof Valvekens et al. (1988).!1 Surface sterilize seeds of Arabidopsis

thaliana (C24) by immersing them asfollows:" 2 min in 70% (v/v) ethanol." 15 min in a solution of 5% (v/v)

N aO Cl and 0.05% (v/v) Tween® 20.!2 Wash seeds 5 times in sterile, distilled

water.

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IV. Pretreatment!1 Remove ethanol from seedlings or flow-

ers (or inflorescences) and transfermaterial to microcentrifuge tubes.

!2 Fix each sample as follows:" Rinse 2 x with 1 ml ethanol." Replace ethanol with 1 ml ethanol/

xylene (1:1) and incubate for 30 min." Rinse 2 x with 1 ml ethanol." Rinse 2 x with 1 ml methanol." Replace methanol with 1 ml of a 1:1

mixture of methanol and [PBTcontaining 1% (v/v) formaldehyde].Rock for 5 min.Note: PBT contains 1 x PBS and0.1% (v / v ) Tween® 20.

!3 Post-fix sample for 25 min in 1 ml PBTcontaining 1% formaldehyde.

!4 Remove fixative and rinse sample with 5 x1 ml PBT.

!5 Wash sample 3 x 1 ml of 2 x SSC (eachwash, 5 min).Note: 1 x SSC contains 150 mM N aCland 15 mM sodium citrate, pH 7.0.

!6 Digest with RN ase A (100 µg/ml in 2 x SSC) for 1 h at 37°C.

!7 Wash 3 x 5 min with 1 ml PBT.!8 Digest with Proteinase K (40 µg/ml in

PBT) for 8 min at 37°C.!9 After the Proteinase K digestion, do the

following:" Rinse 2 x with 1 ml PBT." Wash 2 x 2 min with 1 ml PBT." Rinse 2 x with 1 ml PBT.

!10 Postfix a second time for 25 min with 1 ml PBT containing 1% formaldehyde.

!11 Remove fixative and rinse 5 x with 1 mlPBT.

III. Labeling of the probe DNA!1 Use the following as hybridization

probes:" A mixture of three ribosomal DN A

(rDN A) inserts, each cloned into pBS(I)KS+ (Stratagene, USA) from A.thaliana. The inserts contain the 5.8S,18S and 25S rRN A genes, as well as theintergenic region (IGR) (Unfried andGruendler, 1990; Unfried et al., 1989).

" A 500 bp repeat DN A sequence,cloned into pGem-2 (Promega, USA).The repeat is one of three classes ofhighly repetitive, tandemly arrangedDN A sequences in A. thaliana(Simoens et al., 1988).

!2 Label undigested samples of both probesaccording to the nick translation proce-dures in Chapter IV of this manual. Usethe following labels:" Label the rDN A with either DIG-

dUTP or fluorescein-dUTP.Note: The concentration of substi-tuted nucleotide in the nick transla-tion labeling mixture should be thesame for either fluorescein-dUTP orDIG-dUTP.

" Label the 500 bp repeat DN A withDIG-dUTP.

!3 After nick translation, treat each labeledprobe as follows:" Co-precipitate 1 µg of labeled DN A

with 55 µg of sonicated salmon spermDN A (Sigma, USA).

" Redissolve probe in 25 µl H 2O to aconcentration of 40 ng labeled DN Aper µl.

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VI. Pre-absorption of antibodies (for indirect detection only)!1 Prepare powdered A. thaliana root or

seedling extract as follows:" Grind the root or seedling under liquid

nitrogen." Extract the ground powder with

acetone under liquid nitrogen." Decant the acetone supernatant." Let the residual acetone evaporate

from the precipitate." Use the dry, powdered precipitate in

the pre-absorption procedure below.!2 Dilute each detection antibody, in 4 x SSC

containing 1% (w/v) BSA, to theworking dilution suggested by the manu-facturers and a final volume of 500 µl.

!3 Add approximately 2 mg of powderedA. thaliana root or seedling extract (fromStep 1 above) to the diluted antibody.

!4 Pre-absorb the antibodies overnight at15°C, in the dark.

!5 Centrifuge the pre-absorption mixture.!6 Use the supernatant in the immunocyto-

chemical detection reaction.

VII. Posthybridization washes!1 After overnight hybridization at 37°C

(Procedure V, Step 4), treat the hybridi-zation sample as follows:" Remove the hybridization solution." Wash the sample for 1 h in fresh

hybridization solution at 37°C." Wash the sample 4 x 30 min in hybridi-

zation solution at 37°C." If performing an in situ hybridization

experiment with directly labeled probeDN A (rDN A) in a single labeling exper-iment, proceed to procedure VIII a.

" If performing an in situ hybridizationexperiment requiring indirect detectionwith antibodies, proceed to step 2.

!2 Bring the seedlings or flowers (or inflo-rescences) gradually to 4 x SSC throughthe following washes (all at roomtemperature):" 20 min with a 3:1 mix of hybridization

solution and 4 x SSC." 20 min with a 1:1 mix of hybridization

solution and 4 x SSC." 20 min with a 1:3 mix of hybridization

solution and 4 x SSC." 4 x 5 min with 4 x SSC.

V. In situ hybridization!1 Wash each sample 10 min with 1 ml of a

1:1 mixture of PBT and hybridizationsolution [hybridization solution contains50% formamide (Ultra Pure from USB,USA) in 2 x SSC].

!2 Rinse sample with 2 x1 ml hybridizationsolution.

!3 Remove the hybridization solution andadd the following to each sample (toproduce a final volume of 500 µl ofhybridization solution):" 250 µl formamide." 50 µl 20 x SSC." Enough H 2O to make a total volume,

including probes, of 500 µl." 25 µl of each labeled rDN A probe (for

single or double labeling experiments)." 25 µl labeled 500 bp repeat probe (for

double labeling experiments only).Note: Each labeled probe has a finalconcentration of 2 ng/ µl.

Note: This incubation mixture can beused for either a single label hybridi-zation (fluorescein-labeled probe) withdirect detection; a single label hybridi-zation (DIG-labeled probe) with indirectdetection; or a double label hybridization(both fluorescein- and digoxigenin-labeled probes). See Procedure VIII belowfor details on these different types ofexperiments.

!4 Treat the sample as follows:" Denature target and probe in hybridi-

zation solution for 4 min at 100°C." Place immediately on ice for 3 min." Centrifuge very briefly." Incubate overnight at 37°C to hybridize

probe and target.Caution: I f using a directly labeled(i.e., fluorescent) probe, perform thehybridization incubation and the restof the procedure in the dark .

Table 1: Immunocytochemicaldetection schemes for single anddouble labeling experimentsa All antibodies should be pre-

absorbed, according to ProcedureVI above.

b Fab fragments.c Fab fragments, from Sigma, USA.d Fab fragments.e Whole molecule, from Chemicon,

USA.

VIIIb. Indirect detection!1 After the posthybridization washes (Pro-

cedure VII), remove the 4 x SSC solutionfrom the samples.

!2 Add the first pre-absorbed antibody(from Table 1) to the sample and incubateovernight at 15°C in the dark.

!3 Remove the first antibody and wash thematerial 4 x15 min in 1 ml 4 x SSCcontaining 0.05% (v/v) Tween® 20.

!4 Depending on whether you are using anamplifying (second antibody), do eitherof the following:" I f you use an amplifying antibody,

proceed to Step 5." I f you do not use an amplifying

antibody, proceed to Step 7.!5 If amplifying the signal, wash the sample

4 x 15 min with 4 x SSC containing 1%bovine serum albumin.

!6 Remove the wash solution; add thesecond pre-absorbed antibody (fromTable 1) to the sample, and incubateovernight at 15°C in the dark.

!7 After the last (first or second) antibodyincubation, remove the antibody andwash the sample 4 x 15 min in 1 ml 1 xPBS.

!8 Proceed to the staining and mountingprocedure (Procedure IX).

VIII. Immunocytochemical detectionUse the immunocytochemical detectionschemes detailed in Table 1 to analyze thesingle and double labeling in situ hybridi-zation experiments.

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Antibodya

Type of Experiment Probe Label 1st (detecting) 2nd (amplifying) Procedure to Follow

Single label, rDN A Fluorescein- – – VIIIadirect detection dUTP

Single label, rDN A DIG-dUTP Fluorescein-conju- Fluorescein-conju- VIIIbindirect detection gated anti-DIG anti- gated anti-sheep anti-

body (from sheep)b body (from donkey)c

Double label rDN A Fluorescein- – – VIIIbdUTP

500 bp DIG-dUTP Tetramethylrhoda- Tetramethylrhoda-mine-conjugated mine-conjugatedanti-DIG antibody anti-sheep antibody(from sheep)d body (from rabbit)e

VIIIa. Direct detection

!1 Bring the samples to 1 x PBS graduallythrough the following washes (all at roomtemperature):" 20 min with a 3:1 mix of hybridization

solution and 1 x PBS." 20 min with a 1:1 mix of hybridization

solution and 1 x PBS." 20 min with a 1:3 mix of hybridization

solution and 1 x PBS." 4 x 15 min in 1 ml 1 x PBS.

!2 Proceed to the staining and mountingprocedure (Procedure IX).

#

Xb. Confocal fluorescence microscopy!1 Record images with the MRC-600

Confocal Scanning Laser Microscope(CSLM) System (Bio-Rad, USA). Attachthe CSLM to the DIAPH O T 300inverted microscope fitted with appro-priate lenses (same microscope and lensesas described in Procedure Xa, Step 1above).

!2 Use the K1/K2 filter block combinations(Bio-Rad, USA) and either:" The 568 nm line from a Krypton-

Argon laser (Ion Laser Technology,Utah, USA), to image the propidiumiodide-stained interphase nuclei andthe tetramethylrhodamine-labeledhybrids.

" The 488 nm line from the sameKrypton-Argon laser, to image thefluorescein-labeled hybrids.

Results and discussionFigures 1 and 2 show some of the resultsobtained by FISH of rDN A and the 500 bprepeat on whole mounts of flowers andseedlings from A. thaliana.

The rDN A from A. thaliana covers about5.7 Mbp per haploid genome (Meyerowitzand Pruitt, 1985), and is distributed over twolarge tandem repeats on chromosomes 2 and4 (Maluszynska and H eslop-H arrison, 1991;Murata et al. 1990). Diploid interphasenuclei from A. thaliana exhibit, however, anumber of rDN A-loci ranging from two tomore than four (Bauwens et al. 1991) as canclearly be seen from the merged image inFigure 1.

The 500 bp repeat sequence covers about0.3–0.6 Mbp per haploid genome (Bauwenset al., 1991; Simoens et al., 1988) and exhibits achromosome-specific large cluster (Bauwensand Van O ostveldt, 1991; Bauwens et al.,1991), resulting in two distinct signals indiploid interphase nuclei as can be seen fromthe merged image in Figure 2.

Some helpful remarks should be made aboutconfocal observation of fluorescent signalsin whole mounts.

IX. Staining and mounting!1 Counterstain the sample as follows:

" For single labeling experiments withfluorescein-conjugated antibodies ornucleotides: Counterstain the chromatinof the interphase nuclei with 0.5 µg/mlpropidium iodide (in 1x PBS) for 1 h inthe dark.

" For double labeling experiments withfluorescein- and tetramethylrhodamine-conjugated nucleotides and antibodies:Counterstain the chromatin of theinterphase nuclei with 0.2 µg/ml DAPI(in 1 x PBS) for 1 h in the dark.Note: DAPI is 4,6'-diamidino-2-phenylindole.

!2 Place a few seedlings or flowers (or partof an inflorescence) on a slide.

!3 Apply some tape to the slides to create asupport for the cover slip, so as not tocrush the seedlings or flowers.

!4 Apply a drop of antifade reagent(Vectashield, Vector Laboratories, USA)and cover with a cover slip.

X. Fluorescence microscopy

Xa. Conventional fluorescence microscopy!1 Visually inspect the slides on a

DIAPH O T 300 inverted microscope(N ikon, Japan), fitted with either a 60 x,N A 1.40 oil immersion lens (O lympus,Japan) or an N PL FLUO TAR, 40 x, N A1.30 oil immersion lens (Leitz, FRG).

!2 Use the following filter combinations(Chroma Technology Corp., USA):" To localize propidium iodide-stained

nuclei and tetramethylrhodamine-labeled hybrids: filter block 31014 404.

" To localize DAPI-stained nuclei: filterblock 31000 404.

" To localize fluorescein-labeled hybrids:filter block 31 001 404.

!3 As light source, use a mercury arc lamp(100 W).

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Sequential excitation with the 568 nm andthe 488 nm lines of the Kr-Ar-laser and theK1/K2 filter combinations from the Bio-Rad MRC-600 CSLM, allowed a clearseparation of the red (tetramethylrhoda-mine) and the green (fluorescein) signal. [Seethe signals from the 500 bp repeat (red) andthe rDN A (green) probes in Figure 2.].Especially, the yellow 568 nm line allowsspecific excitation of the red fluorescing dyessuch as tetramethylrhodamine or, preferably,Texas Red™ with almost no excitation offluorescein.

Confocal observation of the preparationsappeared to be absolutely necessary toobtain clear images of the signals, especiallyin the very dense meristematic tissues of theseedling roottips and the developing flowersin the inflorescences. This was especiallytrue for the weaker signal of the 500 bprepeat that, in many cases, could not beobserved through the autofluorescence hazeby conventional fluorescence microscopy.Acquiring digital images through confocalmicroscopy has the added advantage thatimages recorded at different wavelengths canbe easily and accurately merged andcompared.

Finally, the development of FISH protocolson whole mounts for localizing mRN Asequences, as already suggested by deAlmeida Engler et al. (1994), will make itpossible to follow the expression of differentgenes simultaneously at the cellular level, inthree dimensions, through confocalobservation.

ReferencesBauwens, S.; Katsanis, K.; Van Montagu, M.; Van Oostveldt, P.; Engler, G. (1994) Procedure forwhole mount fluorescence in situ hybridization of interphase nuclei on Arabidopsis thaliana.Plant J. 6, 123–131.Bauwens, S.; Van Oostveldt, P. (1991) Cytogeneticanalysis on interphase nuclei with non-isotopic in situ hybridization and confocal microscopy. Med. Fac. Landbouww. Rijksuniv. Gent. 56/3a,753–758.Bauwens, S.; Van Oostveldt, P.; Engler, G.; Van Montagu, M. (1991) Distribution of the rDNAand three classes of highly repetitive DNA in thechromatin of interphase nuclei of Arabidopsis thaliana. Chromosoma 101, 41–48. de Almeida Engler, J.; Van Montagu, M.; Engler, G.(1994) Hybridization in situ of whole-mountmessenger RNA in plants. Plant Mol. Biol. Reporter12, 321–331.

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Figure 1: Whole mount FISH with rDNA on adeveloping flower from A. thaliana. The image isa merged image representing part of the pistil of adeveloping flower, showing fluorescein-labeledrDNA loci (yellow-green) and propidium iodide-stained interphase nuclei (red). An extended focusimage of 15 optical sections through the pistil was created by maximum brightness projectionbefore merging the ‘green’ (rDNA) and ‘red’ (nuclei)images. Bar 25 µm =̂ 17 mm.

Figure 2: Whole mount double labeling FISH withrDNA and the 500 bp repeat on a seedling fromA. thaliana. This merged image represents part ofthe meristematic zone of a seedling roottip. It showsfluorescein-labeled rDNA loci (green) and tetra-methylrhodamine-labeled 500 bp repeat loci (red).An extended focus image of 20 optical sectionsthrough the meristematic zone of the roottip wascreated by maximum brightness projection beforemerging the ‘green’ (rDNA)and ‘red’ (500 bp repeat)images. Bar 25 µm =̂ 15 mm.

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Nederlof, P. M.; van der Ploeg, S.; Wiegant, J.;Raap, A. K.; Tanke, H. J.; Ploem, J. S.; van derPloeg, M. (1990) Multiple fluorescence in situhybridization. Cytometry 11, 126–131.Simoens, C. R.; Gielen, J.; Van Montagu, M.; Inzé, D. (1988) Characterization of highly repetitivesequences of Arabidopsis thaliana. Nucleic AcidsRes. 16, 6753–6766.Tautz, D.; Pfeifle, C. (1989) A nonradioactive in situhybridization method for the localization of specificRNAs in Drosophila embryos reveals translationalcontrol of the segmentation gene hunchback.Chromosoma 98, 81–85.Unfried, I.; Gruendler, P. (1990) Nucleotide sequenceof the 5.8S and 25S rRNA genes and of the internaltranscribed spacers from Arabidopsis thaliana.Nucleic Acids Res. 18, 4011.Unfried, I.; Stocker, U.; Gruendler, P. (1989)Nucleotide sequence of the 18S rRNA gene fromArabidopsis thaliana Co 10. Nucleic Acids Res 7,7513.Valvekens, D.; Van Lijsebettens, M.; Van Montagu,M. (1988) Agrobacterium tumefaciens-mediatedtransformation of Arabidopsis thaliana root explantsby using kanamycin selection. Proc. Natl. Acad. Sci.USA85, 5536–5540.


Lengauer, C.; Speicher, M. R.; Popp, S.; Jauch, A.;Taniwaki, M.; Nagaraja, R.; Riethman, H. C.; Donis-Keller, H.; D’Urso, M.; Schlessinger, D.; Cremer, T. (1993) Chromosomal bar codes producedby multicolor fluorescence in situ hybridization withmultiple YAC clones and whole chromosomepainting probes. Hum. Mol. Gen. 2, 505–512.Ludevid, D.; Höfte, H.; Himelblau, E.; Chrispeels, M. J. (1992) The expression pattern of the tonoplastintrinsic protein !-TIP in Arabidopsis thaliana iscorrelated with cell enlargement. Plant Physiol.100, 1633–1639.Maluszynska, J.; Heslop-Harrison, J. S. (1991)Localization of tandemly repeated DNA sequencesin Arabidopsis thaliana. Plant J. 1, 159–166.Meyerowitz, E. M.; Pruitt, R. E. (1985) Arabidopsisthaliana and plant molecular genetics. Science 229,1214–1218.Murata, M.; Varga, F.; Maluszynska, J.; Gruendler, P.; Schweitzer, D. (1990) Chromosomallocalization of the ribosomal RNA genes in Arabidopsis thaliana by in situ hybridization. In: Schweitzer, D.; Peuker, K.; Loidl, J. (Eds) FourthInternational Conference on Arabidopsis Research,Vienna, Abstracts, 5.

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Reagent Description Cat. No. Pack size DIG-Nick Translation For generation of highly sensitive probes for 1745 816 160 µl Mix* in situ hybridization with digoxigenin-11-dUTP.

Premixed solution for 40 labeling reactionsNick Translation Mix* For generation of highly sensitive probes for 1745 808 200 µl

fluorescence in situ hybridization. The Nick Translation Mix for in situ probes is designed for direct fluorophore-labeling of in situ probes.

Fluorescein-12-dUTP Tetralithium salt, solution, 1 mmol/l 1373 242 25 nmol (25 µl)dNTP Set Set of dATP, dCTP, dGTP, dTTP, lithium salts, 1277 049 1 set,

solutions 4x10 µmol (100 µl)

Anti-Digoxigenin- Fab Fragments from sheep 1207750 200 µg Rhodamine Anti-Digoxigenin- Fab Fragments from sheep 1207741 200 µg FluoresceinTween® 20 1332 465 5x10 mlEGTA Purity: > 98% 1093 053 50 gProteinase K Lyophilizate 161 519 25 mg

745 723 100 mg1000 144 500 mg1092 766 1 g

RNase A Dry powder 109 142 25 mg109 169 100 mg

*This product or the use of this product may be covered by one or more patents of Boehringer MannheimGmbH, including the following: EP patent 0 649 909 (application pending).

References

I NDEXCONTENTS

Cohen, S. M. (1990) Specification of limb develop-ment in the drosophila embryo by positional cuesfrom segmentation genes. Nature 343, 173–177.Corbin, V.; Michelson, A. M.; Abmayr, S. M.; Neel, V.;Alcamo, E.; Maniatis, T.; Young, M. W. (1991) A role for the drosophila neurogenic genes inmesoderm differentiation. Cell Vol. 67, 311–323.Crespo, P.; Angeles-Ros, M.; Ordovas, J. M.;Rodriguez, J. C.; Ortiz, J. M.; Leon, J. (1992) Foamcells from aorta and spleen overexpress apolipo-protein E in the absence of hypercholesterolemia.Biochem. & Biophys. Res. Communications 183 (2),514–523.Crowley, T. E.; Hoey, T.; Liu, J.-K.; Jan, Y. N.; Jan, L. Y.; Tjian, R. (1993) A new factor related to TATA-binding protein has highly restrictedexpression patterns in Drosophila. Nature 361,557–561.Donly, B. C.; Ding, Q.; Tobe, S. S.; Bendena, W. G.(1993) Molecular cloning of the gene for the allato-statin family of neuropeptides from the cockroachDiploptera punctata. Proc. Natl. Acad. Sci. USA90,8807–8811.Doria-Medina, C. L.; Lund, D. D.; Pasley, A.; Sandra, A.; Sivitz, W .I. (1993) Immunolocalizationof GLUT-1 glucose transporter in rat skeletalmuscleand in normal and hypoxic cardia tissue. Am. J.Physiol. 265, E454–E464.Durham, S. K.; Goller, N. L.; Lynch, J. S.; Fisher, S. M.; Rose, P. M. (1993) Endothelin receptor Bexpression in the rat and rabbit lung as determinedby in situ hybridization using nonisotopic probes. J. Cardiovas. Pharm. 22 (Suppl. 8), S1–S3.Fasano, L.; Röder, L.; Coré, N.; Alexandre, E.; Vola,C.; Jacq, B.; Kerridge, S. (1991) The gene teashirt is required for the development of Drosophila embryonic trunk segments and encodes a proteinwith widely spaced zinc finger motifs. Cell 64, 63–79.Frasch, M. (1991) The maternally expressedDrosophila gene encoding the chromatin-bindingprotein BJ1 is a homolog of the vertebrate generegulator of chromatin condensation, RCC1. EMBO J. 10 (5), 1225–1236.

In this chapter general references ofpublications on the use of digoxigenin in insitu hybridization experiments are listedalphabetically to give to the reader anoverview on the wide range of applicationspresently available.

1. mRNA target

A. DNA probea. RPLAida, T.; Yamada, H.; Asano, G. (1990) Expression of type VI procollagen and prolyl-4-hydroxylasemRNA in carbon tetra-chloride induced liver fibrosisstudied by in situ hybridization. Acta Histochem.Cytochem. 23 (6), 817–824.Ayoubi, T. A. Y.; van Duijnhoven, H. L. P.; Coenen, A.J. M.; Jenks, B. G.; Roubos, E. W.; Martens, G. J. M.(1991) Coordinated expression of 7B2 and ! MSH inthe melanotrope cells of Xenopus laevis. Cell TissueRes. 264, 329–334.Bier, E.; Jan, L. Y.; Nung Jan, Y. (1990) Rhomboid, a gene required for dorsoventral axis establishmentand peripheral nervous system development inDrosophila melanogaster. Genes & Development 4,190–203.Bland, Y. S.; Critchlow, M. A.; Ashhurst, D. E. (1991)Digoxigenin as a probe label for in situ hybridizationon skeletal tissue. Histochem. J. 23, 415–418.Blochlinger, K.; Bodmer, R.; Jan, L. Y.; Jan, Y. N.(1990) Patterns of expression of cut, a proteinrequired for external sensory organ development in wild-type and cut mutant Drosophila embryos.Genes & Development 8, 1322–1331.Bochenek, B.; Hirsch, A. (1990) In situ hybridizationof nodulin mRNAs in root nodules using non-radioactive probes. Plant Mol. Biol. Reporter 8 (4),237–248.Breitschopf, H.; Suchanek, G.; Gould, R. M.; Colman,D. R.; Lassmann, H. (1992) In situ hybridization withdigoxigenin-labeled probes: sensitive and reliabledetection method applied to myelinating rat brain.Acta Neuropathol. 84, 581–587.Brown, D. W.; Welsh, R. M.; Like, A. A. (1993)Infection of peripancreatic lymph nodes but notislets precedes kilham rat virus-induced diabetes in BB/Wor rats. J. Virol. 67 (10), 5873–5878.Casanova, J.; Struhl, G. (1993) The torso receptorlocalizes as well as transduces the spatial signalspecifying terminal body pattern in Drosophila. Nature 362, 152–155.Cohen, B.; Wimmer, E. A.; Cohen, S. M. (1991) Early development of leg and wing primordia in the Drosophila embryo. Mechan. Develop. 33,229–240.

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Mlodzik, M.; Baker, N. E.; Rubin, G. M. (1990)Isolation and expression of scabrous, a generegulating neurogenesis in Drosophila. Genes &Development 4, 1848–1861.Nakano, Y.; Guerrero, I.; Hidalgo, A.; Taylor, A.;Whittle, J. R. S.; Ingham, P. W. (1989) A protein with several possible membrane-spanning domainsencoded by the Drosophila segment polarity genepatched. Nature 341, 508–513.Noiri, E.; Kuwata, S.; Nosaka, K.; Tokunaga, K.; Juji, T.; Shibata, Y.; Kurokawa, K. (1994) Tumornecrosis factor-! mRNA Expression in lipopoly-saccharide-stimulated rat kidney. Am. J. Path.144 (6), 1159–1166.Ono, J. K.; McCaman, R. E. (1992) In situhybridization of whole-mounts of Aplysia gangliausing non-radioactive probes. J. Neurosci. Meth.44, 71–79.Radema, S. A.; van Deventer, S. J. H.; Cerami, A.(1991) Interleukin 1" Is expressed predominantlyby enterocytes in experimental colitis. Gastroenterol. 100, 1180–1186.Rao, Yi; Jan, L-Y; Jan, Y. N. (1990) Similarity of theproduct of the Drosophila neurogenic gene big brainto transmembrane channel proteins. Nature 345,163–157.Reuter, R.; Leptin, M. (1994) Interacting functions of snail, twist and huckebein during the early devel-opment of germ layers in Drosophila. Develop. 120,1137–1150.Rothe, M.; Pehl, M.; Taubert, H.; Jäckle, H. (1992)Loss of gene function through rapid mitotic cyclesin the Drosophila embryo. Nature 359, 156–159.Sampedro, J.; Guerrero, I. (1991) Unrestrictedexpression of the Drosophila gene patched allows a normal segment polarity. Nature 353, 187–190.Sano, J.; Miki, K.; Ichinose, M.; Kimura, M.;Kurokawa, K.; Aida, T.; Ishizaki, M.; Asano, G.;Masugi, Y.; Wong, R. N. S.; Takahashi, K. (1989) In situ localization of pepsinogens I and II mRNA inhuman gastric mucosa. Acta Pathol. Japonica. 39(12), 765–771.Schlinger, B. A.; Amur-Umarjee, S.; Shen, P.;Campagnoni, A. T.; Arnold, A. P. (1994) Neuronaland non-neuronal aromatase in primary cultures of developing zebra finch telencephalon. J. Neurosci. 14 (12), 7541–7552.Shermoen, A. W.; O’Farrell, P. H. (1991) Progressionof the cell cycle through mitosis leads to abortion ofnascent transcripts. Cell 67, 303–310.Shin, D. M.; Chiao, P. J.; Sacks, P. G.; Shin, H. J.;Hong, W. K.; Hittelman, W. N.; Tainsky, M. A. (1993)Activation of ribosomal protein S2 gene expressionin a hamster model of chemically induced oralcarcinogenesis. Carcinogenesis 14 (1), 163–166.Sibon, O. C. M.; Cremers, F. F. M.; Boonstra, J.;Humbel, B. M.; Verkleij, A. J. (1993) Localisation ofEGF-receptor mRNA in the nucleus of A431 cells bylight microscopy. Cell Biol. Intern. 17 (1), 1–11.

Fukuda, T.; Nakajima, H.; Fukushima, Y.; Akutsu, I.;Numao, T.; Majima, K.; Motojima, S.; Sato, Y.;Takatsu, K.; Makino, S. (1994) Detection ofinterleukin-5 messenger RNA and interleukin-5protein inbronchial biopsies from asthma bynonradioactive in situ hybridization andimmunohistochemistry. J. Allergy Clin. Immunol.94, 584–593.Grabner, M.; Bachmann, A.; Rosenthal, F.;Striessnig, J.; Schulz, C.; Tautz, D.; Glossmann, H.(1994) Insect calcium channels. Molecular cloningof an !1-subunit from housefly (Musca domestica)muscle. FEBS Letters 339, 189–194.Haenlin, M.; Kramatschek, B.; Campos-Ortega, J. A. (1990) The pattern of transcription of theneurogenic gene delta of Drosophila melanogaster.Develop. 110, 905–914.Heemskerk, J.; DiNardo, S.; Kostriken, R.; O’Farell,P. H. (1991) Multiple modes of engrailed regulationin the progression towards cell fate determination.Nature 352, 404–410.Hülskamp, M.; Pfeifle, C.; Tautz, D. (1990) A morphogenetic gradient of hunchback proteinorganizes the expression of the gap genes krüppeland knirps in the early Drosophila embryo. Nature 346, 577–580.Hukkanen, V.; Heino, P.; Sears, A. E.; Roizman, B.(1990) Detection of Herpes Simplex Virus latency-associated RNA in mouse trigeminal ganglia by insitu hybridization using nonradioactive digoxigenin-labeled DNA and RNA probes. Methods Mol. Cell. Biol. 2, 70–81.Kellerman, K. A.; Mattson, D. M.; Duncan, I. (1990)Mutations affecting the stability of the fushi tarazuprotein of Drosophila. Genes & Development 4,1936–1950.Krauss, S.; Johansen, T.; Korzh, V.; Fjose, A. (1991)Expression pattern of zebrafish pax genes suggest a role in early brain regionalization. Nature 353,267–270.Kumar, G.; Patel, D.; Naz, R. K. (1993) c-MYCmRNAis present in human sperm cells. Cell. & Mol. Biol.Res. 39, 111–117.Levine, P. H.; Jahan, N.; Murari, P.; Manak, M.;Jaffe, E. S. (1992) Detection of human herpesvirus6 in tissues involved by sinus histiocytosis withmassive lymphadenopathy (Rosai-DorfmanDisease). J. Infect. Dis. 166, 291–295.Maggiano, N.; Larocca, L. M.; Piantelli, M.; Ranelletti, F. O.; Lauriola, L.; Ricci, R.; Capelli, A.(1991) Detection of mRNA and hnRNA using adigoxigenin labeled cDNA probe by in situhybridization on frozen tissue sections. Histochem.J. 23, 69–74.Masucci, J. D.; Miltenberger, R. J.; Hoffman, F. M.(1990) Pattern-specific expression of the Drosophiladecapentaplegic gene in imaginal disks is regulatedby 3’cisregulatory elements. Genes & Development4, 2011–2023.

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b. Nick translationCoates, P. J.; Mak, W. P.; Slavin, G.; d’Ardenne, A. J.(1991) Detection of single copies of Epstein-Barrvirus in paraffin wax sections by non-radioactive in situ hybridization. J. Clin. Pathol. 44, 487–491.Dirks, R. W.; Van Dorp, A. G. M.; Van Minnen, J.;Fransen, J. A. M; Van Der Ploeg, M.; Raap, A. K.(1992) Electron microscopic detection of RNAsequences by non-radioactive in situ hybridizationin the mollusk lymnaea stagnalis. J. Histochem.Cytochem. 40 (11), 1647–1657.Dirks, R. W.; Van de Rijke, F. M.; Fujishita, S.; VanDer Ploeg, M.; Raap, A. K. (1993) Methodologies forspecific intron and exon RNA localization in culturedcells by haptenized and fluorochromized probes. J. Cell Sci. 104, 1187–1197.Flemming, K. A.; Evans, M.; Ryley, K. C.; Franklin,D.; Lovell-Badge, R. H.; Morey, A. L. (1992)Optimization of non-isotopic in situ hybridization onformalin-fixed, paraffin-embedded material usingdigoxigenin-labeled probes and transgenic tissues.J. Pathol. 167, 9–17.Whawell, S. A.; Wang, Y.; Fleming, K. A.; Thompson,E. M.; Thompson, J. N. (1993) Localization ofplasminogen activator inhibitor-1 production ininflamed appendix by in situ mRNA hybridization. J. Pathol. 169, 67–71.

c. cDNA synthesisUltsch, A.; Schuster, C. M.; Laube, B.; Schloss, P.;Schmitt, B.; Betz, H. (1992) Glutamate receptors of Drosophila melanogaster: Cloning of a kainate-selective subunit expressed in the central nervoussystem. Proc. Natl. Acad. Sci. USA89, 10484–10488.

d. PCRBarka, T.; van der Noen, H. (1993) Expression of thecysteine proteinase inhibitor cystatin C gene in ratheart: Use of digoxigenin-labeled probes generatedby polymerase chain reaction directly for in situand northern blot hybridizations. J. Histochem.Cytochem. 41 (12) 1863–1867. Barka, T.; van der Noen, H. (1994) Expressions ofthe genes for cysteine proteinase inhibitors cystatinC and cystatin S in rat submandibular salivarygland. Archs oral Biol. 39 (4), 307–314.Barka, T.; van der Noen, H. (1994) Expression of thecysteine proteinase inhibitor cystatin C mRNA in rateye. Anatom. Rec. 239, 343–348.Cripe, L.; Morris, E.; Fulton, A. B. (1993) VimentinmRNAlocationchangesduringmuscledevelopment.Proc. Natl. Acad. Sci. USA90, 2724–2728.Dirks, R. W.; van de Rijke, F. M.; Fujishita, S.; vander Ploeg, M.; Raap, A. K. (1993) Methodologies forspecific intron and exon RNA localization in culturedcells by haptenized and fluorochromized probes. J. Cell Sci. 104, 1187–1197.

Sommer, R.; Tautz, D. (1991) Nonradioactive in situhybridization to sectioned tissues. Trends Genet. 7(4), 110.Steinmetz, R. W.; Grant, A. L.; Malven, P. V. (1993)Transcription of prolactin gene in milk secretorycells of the rat mammary gland. J. Endocrinol. 136,271–276.Tautz, D.; Pfeifle, C. (1989) A non-radioactive in situhybridization method for the localization of specificRNAs in Drosophila embryos reveals translationalcontrol of the segmentation gene hunchback.Chromosoma (Berl.) 98, 81–85.Treacy, M. N.; He, X.; Rosenfeld, M. G. (1991) I-POU:a POU-domain protein that inhibits neuron-specificgene activation. Nature 350, 577–584.Vassias, I.; Perol, S.; Coulombel, L.; Thebault, M.-C.; Lagrange, P. H.; Morinet, F. (1993) An in situhybridization technique for the study of B19 humanparvovirus replication in bone marrow cell cultures.J. Virol. Meth. 44, 329–338.Wiethege, Th.; Voss, B.; Pohle, Th.; Fisseler-Eckhoff,A.; Müller, K. M. (1991) Localization of elastase andtumor necrosis factor-! mRNA by non-radioactive in situ hybridization in cultures of alveolarmacrophages. Path. Res. Pract. 187, 912–915.Wu, T.-C.; Mann, R. B.; Epstein, J. I.; MacMahon, E.;Lee, W. A.; Charache, P.; Hayward, S. D.; Kurman, R. J.; Hayward, G. S.; Ambinder, R. F. (1991) Abundant expression of EBER 1 small nuclear RNA in nasopharyngeal carcinoma. Amorphologicallydistinctive target for detection of Epstein-Barr virusin formalin-fixed paraffin-embedded carcinomaspecimens. Am. J. Pathol. 128 (6), 1461–1469.Wulf, M.; Bosse, A.; Wiethege, T.; Voss, B.; Müller,K. M. (1994) Localization of collagen types I, II andIII mRNAs in human heterotopic ossification by non-radioactive in situ hybridization. Path. Res.Pract. 190, 25–32.Xing, Y.; Johnson, C. V.; Dobner, P. R.; Lawrence, J. B. (1993) Higher level organization of individualgene transcription and RNA splicing. Science 259,1326–1330.Yamada, H.; Aida, T.; Taguchi, K.; Asona, G. (1989)Localization of type III procollagen mRNA in areas of liver fibrosis by in situ hybridization. Acta Pathol.Japonica. 39 (11), 719–724.Zhang, P.; Knowles, B. A.; Goldstein, L. S. B.;Hawley, R. S. (1990) A kinesin-like protein requiredfor distributive chromosome segregation inDrosophila. Cell 62, 1053–1062.Zhao, T.-J.; Chiu, T. H.; Rosenberg, H. C. (1994)Reduced expression of #-Aminobutyric acid typeA/benzodiazepine receptor #2 and !5 subunitmRNAs in brain regions of flurazepam-treated rats.Mol. Pharmacol. 45, 657–663.Zheng, M. H.; Fan, Y.; Wysocki, S.; Wood, D. J.;Papadimitriou, J. M. (1993) Detection of mRNA forcarbonic anhydrase II in human oesteoclast-likecells by in situ hybridization. J. Bone Mineral Res. 8(1), 113–118.

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Birk, P. E.; Grimm, P. C. (1994) Rapid nonradioactivein situ hybridization for interleukin-2 mRNA withriboprobes generated using the polymerase chainreaction. J. Immunol. Meth. 167, 83–89.Bochenek, B.; Hirsch, A. (1990) In situ hybridizationof nodulin mRNAs in root nudules using non-radioactive probes. Plant Mol. Biol. Reporter 8 (4),237–248.Brewer, L. M.; Sheardown, S. A.; Brown, N. A.(1993) HMG-CoA reductase mRNA in the post-implantation rat embryo studied by in situhybridization. Tetratol. 47, 137–146.Broide, D. H.; Paine, M. M.; Firestein, G. S. (1992)Eosinophils express interleukin 5 and granulocytemacrophage-colony-stimulating factor mRNA atsites of allergic inflammation in asthmatics. J. Clin. Invest. 90, 1414–1424.Bromley, L.; McCarthy, S. P.; Stickland, J. E.; Lewis, C. E.; McGee, J. (1994) Non-isotopic in situdetection of mRNA for interleukin-4 in archivalhuman tissue. J. Immunol. Meth. 167, 47–54.Brown, P. A. J.; Wilson, H. M.; Reid, F. J.; Booth, N.A.; Simpson, J. G.; Morrison, L.; Power, D. A.;Haites, N. E. (1994) Urokinase-plasminogenactivator is synthesized in vitro by humanglomerular epithelial cells but not by mesangialcells. Kidney Int. 45, 43–47.Bu, G.; Maksymovitch, E. A.; Nerbonne, J. M.;Schwartz, A. L. (1994) Expression and function ofthe low density lipoprotein receptor-related protein(LRP) in mammalian central neurons. J. Biol. Chem.269 (28), 18521–18528.Butcher, L. L.; Oh, J. D.; Woole, N. J.; Edwards, R. H.; Roghani, A. (1992) Organization of centralcholinergic neurons revealed by combined in situhybridization histochemistry and choline-O-acetyltransferase immunocytochemistry.Neurochem. Int. 21 (3), 429–445.Cai, W. Q.; Terenghi, G.; Bodin, P.; Burnstock, G.;Polak, J. M. (1993) In situ hybridization of atrialnatriuretic peptide mRNA in the endothelial cells of human umbilical vessels. Histochem. 100,277–283. Campbell, A. M.; Wuytack, F.; Fambrough, D. M.(1993) Differential distribution of the alternativeforms of the sarcoplasmic/endoplasmic reticulumCa2+-ATPase, SERCA2b and SERCA2a, in the avianbrain. Brain Res. 605, 67–76.Canas, L. A.; Busscher, M.; Angenent, G. C.;Beltran, J.-P.; van Tunen, A. J. (1994) Nuclearlocalization of the petunia MADS box protein FBP1.Plant J. 6 (4), 597–604.Caroni, P.; Schneider, C. (1994) Signaling by insulin-like growth factors in paralyzed skeletal muscle:rapid induction of IGF-1 expression in muscle fibersand prevention of interstitial cell proliferation byIGF-BP5 and IGF-BP4. J. Neurosci. 14 (5),3378–3388.

Hannon, K.; Johnstone, E.; Craft, L. S.; Little, S. P.;Smith, C. K.; Heiman, M. L.; Santerre, R. F. (1993)Synthesis of PCR-derived, single-stranded DNAprobes suitable for in situ hybridization. Anal.Biochem. 212, 421–427.Iwamura, M.; Wu, G.; Abrahamsson, P.-A.; DiSant’Agnese, P. A.; Cockett, A. T. K.; Deftos, L. J.(1994) Parathyroid hormone-related protein isexpressed by prostatic neuroendocrine cells.Urology 43 (5), 667–674.Räsänen, L. A.; Karvonen, U.; Pösö, A. R. (1993)Localization of xanthine dehydrogenase mRNA inhorse skeletal muscle by in situ hybridization withdigoxigenin-labeled probe. Biochem. J. 292,639–641.Sommer, R. J.; Tautz, D. (1993) Involvement of anorthologue of the Drosophila pair-rule gene hairy insegment formation of the short germ-band embryoof Tribolium (Coleoptera). Nature 361, 448–450.Yang, X.; Stedra, J.; Cerny, J. (1994) Repertoire diversity of antibody response to bacterial antigensin aged mice. IV. Study of VH and VL gene utilizationin splenic antibody foci by in situ hybridization. J. Immunol. 152, 2214–2220.

B. RNA probesAdham, I. M.; Burkhardt, E.; Benahmed, M.; Engel,W. (1993) Cloning of a cDNA for a novel insulin-likepeptide of the testicular leydig cells. J. Biol. Chem.268 (35), 26668–26672.Ahn, K. Y.; Madsen, K. M.; Tisher, C. C.; Kone, B. C.(1993) Differential expression and cellulardistribution of mRNAs encoding !- and "-isoformsof Na+-K+-ATPase in rat kidney. Am. J. Physiol. 265,792–801.Bachmann, S.; Le Hir, M.; Eckardt, K.-U. (1993) Co-localization of erythropoietin mRNA and ecto-5’-nucleotidase immunoreactivity in peritubular cellsof rat renal cortex indicates that fibroblasts produceerythropoietin. J. Histochem. Cytochem. 41,335–341.Baldino, F.; Robbins, E.; Grega D.; Meyers, S. L.;Springer, J. E.; Lewis, M. E. (1989) Non-radioactivedetection of NGF-receptor messenger RNA withdigoxigenin-UTP labeled RNA probes. 19th AnnualMeeting Soc. Neuroscience, Phoenix, Arizona, USA,Oct. 29–Nov. 3, 1989. Soc. Neurosci. Abst.15 (1), 864.Barthel, L. K.; Raymond, P. A. (1993) Subcellularlocalization of !-tubulin and opsin mRNA in thegoldfish retina using digoxigenin-labeled cRNAprobes detected by alkaline phosphatase and HRPhistochemistry. J. Neurosci. Meth. 50, 145–152.Biffo, S.; Verdun di Cantogno, L.; Fasolo, A. (1992)Double labeling with non-isotopic in situ hybrid-ization and BrdU immunohistochemistry:Calmodulin (CaM) mRNA expression in post-mitoticneurons of the olfactory system. J. Histochem.Cytochem. 40 (4), 535–540.

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Wu, T.-C.; Lee, W. A.; Pizzorno, M. C.; Au, W.-C.;Chan, Y.-J.; Hruban, R. H.; Hutchins, G. M.;Hayward, G. S. (1992) Localization of the humancytomegalovirus 2.7-kb major early "-genetranscripts by RNA in situ hybridization inpermissive and nonpermissive infections. Am. J.Pathol. 141, 1247–1254.Wu, T.-C.; Pizzorno, M. C.; Hayward, G. S.;Willoughby, S.; Neumann, D. A.; Rose, N. R.; Ansari, A. A.; Beschorner, W. E.; Baughman, K. L.;Herskowitz, A. (1992) In situ detection of humancytomegalovirus immediate-early gene transcriptswithin cardiac myocytes of patients with HIV-associated cardiomyopathy. AIDS6, 777–785.Wu, T.-C.; Kuo, T.-T. (1993) Study of epstein-barrvirus early RNA 1 (EBER 1) expression by in situhybridization in thymic epithelial tumors of chinesepatients in Taiwan. Hum. Pathol. 24, 235–238.Xu, X.-C.; Ro, J. Y.; Lee, J. S.; Shin, D. M.; Hong, W. K.; Lotan, R. (1994) Differential expression ofnuclear retinoid receptors in normal, premalignant,and malignant head and neck tissues. Cancer Res.54, 3580–3587.Xu, X.-C.; Clifford, J. L.; Hong, W. K.; Lotan, R.(1994) Detection of nuclear retinoic acid receptormRNA in histological tissue sections using non-radioactive in situ hybridization histochemistry.Diagn. Mol. Pathol. 3 (2), 122–131.Yan, Y.; Lagenaur, C.; Narayanan, V. (1993)Molecularcloning of M6: identification of a PLP/DM20 genefamily. Neuron 11, 423–431.Ying, S.; Durham, S. R.; Barkans, J.; Masuyama, K.;Jacobson, M.; Rak, S.; Löwhagen, O.; Moqbel, R.;Kay, A. B.; Hamid, Q. A. (1993) T cells are theprincipal source of Interleukin-5 mRNA in allergen-induced rhinitis. Am. J. Respir. Cell Mol. Biol. 9,356–360.Yokouchi, Y.; Sasaki, H.; Kuroiwa, A. (1991)Homeobox gene expression correlated with thebifurcation process of limb cartilage development.Nature 353, 443–445.Yoshimura, A.; Gordon, K.; Alpers, C. E.; Floege, J.;Pritzl, P.; Ross, R.; Couser, W. G.; Bowen-Pope, D. F.; Johnson, R. J. (1991) Demonstration of PDGFB-chainmRNAinglomeruli inmesangial proliferativenephritis by in situ hybridization. Kidney Internat.40, 470–476.Young, I. D.; Stewart, R. J.; Ailles, L.; Mackie, A.;Gore, J. (1993) Synthesis of digoxigenin-labeledcRNA probes for nonisotopic in situ hybridizationusing reverse transcription polymerase chainreaction. Biotechn. Histochem. 68 (3), 153–158.Zhou, X.; Sasaki, H.; Lowe, L.; Hogan, B. L. M.;Kuehn, M. R. (1993) Nodal is a novel TGF-"-like gene expressed in the mouse node duringgastrulation. Nature 361, 543–547.Zurbriggen, A.; Müller, C.; Vandevelde, M. (1993) In situ hybridization of virulent canine distempervirus in brain tissue, using digoxigenin-labeled probes. Am. J. Vet. Res. 54 (9), 1457–1461.

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Harper, S. J.; Pringle, J. H.; Gillies, A.; Allen, A. C.;Layward, L.; Feehally, J.; Lauder, I. (1992) Simul-taneous in situ hybridization of native mRNA andimmunoglobulin detection by conventionalimmunofluorescence in paraffin way embeddedsections. J. Clin. Pathol. 45, 114–119.Harvie, R.; Elvin, P.; McArdle, C.; Morten, J. E. N.;McNicol, A. M. (1991) Detection of mRNA for riboso-mal phosphoprotein P2 in normal colon and colonictumours by in situ hybridization. J. Pathol. 164,67–73.Hilton, D. A.; Day; C.; Pringle, J. H.; Fletcher, A.; Cham-bers, S. (1992) Demonstration of the distribution ofCoxsackievirusRNAinneonatal mice by non-isotop-ic in situ hybridization. J. Virol. Meth. 40, 155–162.Hilton, D. A.; Fletcher, A.; Pringle, J. H. (1994)Absence of coxsacki viruses in idiopathicinflammatory muscle disease by in situ hybridi-zation. Neuropathol. Appl. Neurobiol. 20, 238–242.Humpel, C.; Lindqvist, E.; Olson, L. (1993) Detectionof nerve growth factor mRNA in rodent salivaryglands with digoxigenin- and 33P-labeled oligo-nucleotides: effects of castration and sympa-thectomy. J. Histochem. Cytochem. 41 (5), 703–708.Kendall, C. H.; Roberts, P. A.; Pringle, J. H.; Lauder, I. (1991) The expression of parathyroidhormone messenger RNA in normal and abnormalparathyroid tissue. J. Pathol. 165, 111–118.Kennedy, R. E.; Hutchins, J. B. (1992)Cholineacetyl-transferase expression studied with an oligonucleo-tide probe. Cell. Mol. Neurobiol. 12 (4), 309–315.Khan, G.; Coates, P .J.; Kangro, H. O.; Slavin, G.(1992)EpsteinBarr virus (EBV) encoded small RNAs:Targets for detection by in situ hybridization witholigonucleotideprobes. J. Clin. Pathol. 45, 616–620.Khan, G.; Norton, A. J.; Slavin, G. (1993) Epstein-Barr virus in angioimmunoblastic T-cell lymphomas.Histophatol. 22, 145–149.Kirchgessner, A. L.; Liu, M.-T.; Howard, M. J.;Gershon, M. D. (1993) Detection of the 5-HT1AReceptor and 5-HT1A Receptor mRNA in the ratbowel and pancreas: comparison with 5-HT1PReceptors. J. Comparative Neurol. 327, 233–250.Koji, T.; Brenner, R. M. (1993) Localization ofestrogen receptor messenger ribonucleic acid inrhesus monkey uterus by nonradioactive in situhybridization with digoxigenin-labeled oligodeoxy-nucleotides. Endocrinol. 132, 382–392.Komminoth, P. (1992) Digoxigenin as an alternativeprobe labeling for in situ hybridization. Diagn. Mol.Pathol. 1 (2), 142–150.Komminoth, P.; Merk, F. B.; Leav, I.; Wolfe, H. J.;Roth, J. (1992) Comparison of 35S- and digoxigenin-labeled RNA and oligonucleotide probes for in situhybridization. Histochem. 98, 217–228.Krizbai, I.; Deli, M.; Lengyel, I.; Maderspach, K.;Pákáski, M.; Joó, F.; Wolff, J. R. (1993) In situhybridization with digoxigenin labeled oligonucleo-tide probes: detection of CAMK-II gene expressionin primary cultures of cerebral endothelial cells.Neurobiol. 1 (3), 235–240.

Dickerson, D. S.; Huerter, B. S.; Morris, S. J.;Chronwall, B. M. (1994) POMC mRNA levels inindividual melanotropes and GFAP in glial-like cellsin rat pituitary. Peptides 15 (2), 247–256.Dirks, R. W.; Van Dorp, A. G. M.; Van Minnen, J.;Fransen, J. A. M.; Van der Ploeg, M.; Raap, A. K.(1992) Electron microscopic detection or RNAsequences by non-radioactive in situ hybridizationin the mollusk lymnaea stagnalis. J. Histochem.Cytochem. 40, 1647–1657.Dirks, R. W.; Van Gijlswijk, R. P. M.; Vooljs, M. A.;Smit, A. B.; Bogerd, J.; Van Minnen, J.; Raap, A. K.;Van der Ploeg, M. (1991) 3’End fluorochromized andhaptenized oligonucleotides as in situ hybridizationprobes for multiple, simultaneous RNA detection.Experim. Cell Res. 194, 310–315.Dirks, R. W.; Van der Rijke, F. M.; Fujishita, S.; Van der Ploeg, M.; Raap, A. K. (1993) Methodologiesfor specific intron and exon RNA localization incultured cells by haptenized and fluorochromizedprobes. J. Cell Sci. 104, 1187–1197.Dumas, S.; Horellou, P.; Helin, C.; Mallet, J. (1992)Co-expression of tyrosine hydroxylase messengerRNA1 and 2 in human ventral mesencephalonrevealed by digoxigenin- and biotin-labeled oligo-deoxyribonucleotides. J. Chem. Neuroanatomy 5,11–18.Crabb, I. D.; Hughes, S. S.; Hicks, D. G.; Puzas, J. E.;Tsao, G. J. Y.; Rosier, R. N. (1992) Nonradioactive insituhybridizationusingdigoxigenin-labeled oligo-nucleotides. Am. J. Pathol. 141 (3), 579–589.Farquharson, M.; Harvie, R.; McNicol, A. M. (1990)Detection of messenger RNA using a digoxigeninend labeled oligonucleotide probe. J. Clin Pathol.43, 424–428.Farquharson, M.; Harvie, R.; McNicol, A. M. (1990)Detection of pro-opiomelanocortin messenger RNAusingadigoxigeninend labeledoligodeoxynucleotideprobe. J. Endocrinol. 124 (Supplement).Fevre-Montange, M.; Trembleau, A.; Landry, M.;Calas, A. (1992) Hybridation in situ avec des sondesoligonucléotidiques marquées à la digoxigénine:application à la détection des ARNm de la vaso-pressine et de l’ocytocine. Techniques, 479–486.Grega, D.; Cavanaugh, T.; Martin, R.; Lewis, M.;Baldino, F. (1989) Insituhybridizationhistochemistryusinganew non-radioactive detection method. ICSUShort Reports, Advances inGeneTechnology: Molec-ular NeurobiologyandNeuropharmacology, Proceed-ings of the 1989 Miami Bio/Technology WinterSymposium,Vol. 9, p. 69, IRLPress, McLean, Virginia.Guellec Le, P.; Dumas, S.; Volle, G. E.; Pidoux, E.;Moukhtar, M. S.; Treilhou-Lahille, F. (1993) An efficient method todetect calcitoninmRNAinnormaland neoplastic rat c-cells (Medullary ThyroidCarcinoma) by in situ hybridization using adigoxigenin-labeled syntheticoligodeoxyribonucleotide probe. J. Histochem. Cytochem. 41, 389–395. Hamada, K.; Okawara, Y.; Fryer, J. N.; Tomonaga,A.; Fukuda, H. (1994) Localization of mRNA ofprocollagen !1 type I in torn supraspinatus tendons.Clin. Orthopaed. Related Res. 304, 18–21.

8. Miscellaneous

Schöfer, C.; Müller, M.; Leitner, M. D.; Wachtler, F.(1993) The uptake of uridine in the nucleolus occursin the dense fibrillar component. Cytogenet. CellGenet. 64, 27–30.Wansink, D. G.; Manders, E. E. M.; van der Kraan, I.;Aten, J. A.; van Driel, R.; de Jong, L. (1994) RNApolymerase II transcription is concentrated outsidereplication domains throughout S-phase. J. Cell Sci.107, 1449–1456.

200

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7. Apoptosis research using DIG-dUTP and terminaltransferase

Billig, H.; Furuta, I.; Hsueh, A. J. (1993) Estrogensinhibit and androgens enhance ovarian granulosacell apoptosis. Endocrinol. 133 (5) 2204–2212.Billig, H.; Furuta, I.; Hsueh, A. J. W. (1994) Gonado-tropin-releasing hormone directly induces apoptoticcell death in the rat ovary: Biochemical and in situdetection of deoxyribonucleic acid fragmentation ingranulosa cells. Endocrinol. 134 (1), 245–252.Gold; R.; Schmied, M.; Rothe, G.; Zischler, H.;Breitschopf, H.; Wekerle, H.; Lassmann, H. (1993)Detection of DNA fragmentation in apoptosis:Application of in situ nick translation to cell culturesystems and tissue sections. J. Histochem.Cytochem. (in press).Gorczyca, W.; Bigman, K.; Mittelman, A.; Ahmed, T.;Gong, J.; Melamed, M. R.; Darzynkiewicz, Z. (1993)Induction of DNA strand breaks associated withapoptosis during treatment of leukemias. Leukemia7 (5), 659–670.

Gorczyca, W.; Gong, J.; Ardelt, B.; Traganos, F.;Darzynkiewicz, Z. (1993) The cell cycle relateddifferences in susceptibility of HL-60 cells toapoptosis induced by various antitumor agents.Cancer Res. 53, 3186–3192.Gorczyca, W.; Gong, J.; Darzynkiewicz, Z. (1993)Detection of DNA strand breaks in individualapoptotic cells by the in situ terminal deoxynucleo-tidyl transferase and nick translation assays. Canc.Res. 53, 1945–1951.Sgonc, R.; Boeck, G.; Dietrich, H.; Gruber, J.;Reicheis, H.; Wick, G. (1994) Simultaneous deter-mination of cell surface antigens and apoptosis. TIG10 (2), 41–42.

Myatt, N.; Coghill, G.; Morrison, K.; Jones, D.; Cree, I. A. (1994) Detection of tumour necrosisfactor-! in sarcoidosis and tuberculosisgranulomas using in situ hybridization. J. Clin.Pathol. 47, 423–426.Nakagawa, M.; Kukita, T.; Nakasima, A.; Kurisu, K.(1994) Expression of the type I collagen gene in ratperiodontal ligament during tooth movement asrevealed by in situ hybridization. Archs oral Biol. 39(4), 289–294.Negro, F.; Papotti, M.; Pacchioni, D.; Galimi, F.;Bonino, F.; Bussolati, G. (1994) Detection of humanandrogen receptor mRNA in hepatocellularcarcinoma by in situ hybridization. Liver 14,213–219.Nicoloso, M.; Caizergues-Ferrer, M.; Michot, B.;Azum, M.-C.; Bachellerie, J.-P. (1994) U20, a novelsmall nucleolar RNA, is encoded in an intron of thenucleolin gene in mammals. Mol. Cell. Biol. 14 (9),5766–5776.O’Keefe, R. J.; Puzas, J. E.; Loveys, L.; Hicks, D. G.;Rosier, R. N. (1994) Analysis of type II and type Xcollagensynthesis inculturedgrowthplatechondro-cytes by in situ hybridization: rapid induction oftype X collagen in culture. J. Bone Mineral Res. 9(11), 1713–1722.Ong, W. Y.; Garey, L. J.; Sumi, Y. (1994) Distributionof preprosomatostatin mRNA in the rat parietal andtemporal cortex. Mol. Brain Res. 23, 151–156.Pacchioni, D.; Papotti, M.; Andorno, E.; Bonino, F.;Mondardini, A.; Oliveri, F.; Brunetto, M.; Ussolati,G.; Negro, F. (1993) Expression of estrogen receptormRNA in tumorous and non-tumorous liver tissue asdetected by in situ hybridization. J. Surgical Oncol.Suppl. 3, 14–17.Papotti, M.; Pacchioni, D.; Negro, F.; Bonino, F.;Bussolati, G. (1994) Albumin gene expression inliver tumors: diagnostic interest in fine needleaspiration biopsies. Modern Pathol. 7 (3), 271–275.Paulmyer-Lacroix, O.; Anglade, G.; Grino, M. (1994)Insulin-induced hypoglycaemia increase colocaliza-tion of corticotrophin-releasing factor and argininevasopressin mRNAs in the rat hypothalamicparaventricular nucleus. J. Mol. Endocrinol. 13,313–320.Samoszuk, M.; Nansen, L. (1990) Detection ofInterleukin-5 messenger RNA in Reed-Sternbergcells of Hodgkin’s desease with eosinophilia. Blood 75 (1), 13–16.Sharara, F. I.; Nieman, L. K. (1994) Identificationandcellular localization of growth hormone receptorgene expression in the human ovary. J. Clin.Endocrinol. Metabolism 79 (2), 670–672.Shorrock, K.; Roberts, P.; Pringle, J. H.; Lauder, I.(1991)Demonstration of insulin and glucagon mRNAin routinely fixed and processed pancreatic tissueby in situ hybridization.J. Pathol. 165, 105–110.Thomas, G. A.; Davies, H. G.; Williams, E. D. (1994)Site of production of IGF1 in the normal andstimulated mouse thyroid. J. Pathol. 173, 355–360.

Krusche, C.; Beier, H. M. (1994) Localization ofuteroglobin mRNA by nonradioactive in situhybridization in the pregnant rabbit endometrium.Ann. Anat. 176, 23–31.Laverdure, A.-M.; Carette-Desmoucelles, C.;Breuzet, M.; Descamps, M. (1994) Neuropeptidesand related nucleic acid sequences detected inpeneid shrimps by immunohistochemistry andmolecular hybridizations. Neurosci. 60 (2), 569–579.Laverdure, A.-M.; Breuzet, M.; Soyez, D.; Becker, J.(1992) Detection of the mRNA encoding vitellogene-sis inhibiting hormone in neurosecretory cells of theX-organ in homarus americanus by in situ hybridi-zation. Gen. Compar. Endocrinol. 87, 443–450.Lee, D.; Huang, W.; Yang, Z.; Copolov, D. L.; Lim, A. T. (1992) In vitro evidence for modulation ofmorphological and functional development ofhypothalamic immunoreactive atrial natriureticpeptide neurons by cyclic 3', 5'-adenosinemonophosphate. Endocrinol. 131, 911–918.Lewis, M. E.; Robbins, E.; Grega, D.; Baldino, F.(1989) Nonradioactive detection of vasopressionand somatostatin mRNA with Digoxigenin-labeledoligonucleotide probes. Ann. NY Acad. Sci. V:Molecular Biology: The Future of NeuropeptideResearch, pp 246–253.Matute, C.; Wahle, P.; Gutiérrez-Igarza, K.; Albus, K.(1993) Distribution of neurons expression substanceP receptor messenger RNA in immature and adultcat visual cortex. Exp. Brain Res. 97, 295–300.Mertani, H. C.; Waters, M. J.; Jambou, R.; Gossard, F.;Morel, G. (1994) Growth hormone receptor indingprotein in rat anterior pituitary. Neuroendocrinol. 59,483–494.Miranda, R. C.; Toran-Allerand, C. D. (1992)Developmental expression of estrogen receptormRNA in the rat cerebral cortex: A nonisotopic insitu hybridization histochemistry study. CerebralCortex Jan/Feb 2, 1–15.Mitchel, V.; Gambiez, A.; Beauvillain, J. C. (1993)Fine-structural localization of proenkephalin mRNAsin the hypothalamic magnocellular dorsal nucleusof the guinea pig: a comparison of radioisotopic andenzymatic in situ hybridization methods at the light-and electron-microscopic levels. Cell Tissue Res.274, 219–228.Millar, M. R.; Sharpe, R. M.; Maguire, S. M.;Saunders, P. T. K. (1993) Cellular localisation ofmessenger RNAs in rat testis: application ofdigoxigenin-labeled ribonucleotide probes toembedded tissue. Cell Tissue Res. 273, 269–277.Miyazaki, M.; Nikolic-Paterson, D. J.; Masayuki, E.;Nomoto, Y.; Sakai, H.; Atkins, R. C.; Koji, T. (1994) A sensitive method of non-radioactive in situhybridization for mRNA localization within humanrenal biopsy specimens: use of digoxigenin labeledoligonucleotides. Int. Med. 33, 87–91.Murray, G. I.; Paterson, P. J.; Ewen, S. W. B.; Melvin,W. T. (1992) In situ hybridization of albumin mRNAin normal liver and hepatocellular carcinoma with adigoxigenin labeled oligonucleotide probe. J. Clin.Pathol. 45, 21–24.

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Yamada, S.; Takahashi, M.; Hara, M.; Sano, T.; Aiba,T.; Shishiba, Y.; Suzuki, T.; Asa, S. L. (1994) Growthhormone and prolactin gene expression in humandensely and sparsely granulated somatotrophadenomas by in situ hybridization with digoxigenin-labeled probes. Diagn. Mol. Pathol. 3 (1), 46–52.Young, W. S. III (1989) Simultaneous use ofdigoxigenin- and radiolabeled oligodeoxy-ribonucleotide probes for hybridization histo-chemistry. Neuropeptides (Edinburgh) 18, 271–275.Young, W. S. III; Horvath, S.; Palkovits, M. (1990)The influence of hyperosmolality and synapticinputs on galanin and vasopressin expression in thehypothalamus. Neurosci. 39 (1), 115–125.Young, W. S. III; Reynolds, K.; Shepard, E. A.;Gainer, H.; Castel, M. (1990) Cell-specific expressionof the rat oxytocin gene in transgenic mice. J. Neuroendocrinol. 2 (6), 917–925.Zupanc, G. K. H.; Okawara, Y.; Zupanc, M. M.; Fryer, J. N.; Maler, L. (1991) In situ hybridization ofputative somatostatin mRNA in the brain of electricgymnotiform fish. Neuro Report 2, 707–710.

c. Chemical synthesisHassall, C. J. S.; Stanford, S. C.; Burnstock, G.;Buckley, N. J. (1993) Co-expression of fourmuscarinic receptor genes by the intrinsic neuronsof the rat and guinea-pig heart. Neurosci. 56 (4),1041–1048.Hell, K.; Pringle, J. H.; Hansmann, M.-L.; Lorenzen,J.; Colloby, P.; Lauder, I.; Fischer, R. (1993)Demonstration of light chain mRNA in hodgkin’sdisease.J. Pathol. 171, 137–143.Hodges, E.; Howell, W. M.; Tyacke, S. R.; Wong, R.;Cawley, M. I. D.; Smith, J. L. (1994) Detection of T-cell receptor " chainmRNAin frozen and paraffin-embedded biopsy tissue using digoxigenin-labeledoligonucleotide probes in situ. J. Pathol. 174,151–158.Li, D.; Bell, J.; Brown, A.; Berry, C. L. (1994) Theobservationof angiogeninandbasic fibroblastgrowth factor gene expression in human colonicadenocarcinomas, gastric adenocarcinomas, andhepatocellular carcinomas. J. Pathol. 172, 171–175.Shanks, J. H.; Lappin, T. R. J.; Hill, C. M. In situhybridization for erythropoietin messenger RNAusing digoxigenin-labeled oligonucleotides. AnnalsNew York Academy of Sciences.Thomas, G. A.; Neonakis, E.; Davies, H. G.; Wheeler,M. H.; Williams, E. D. (1994) Synthesis and storagein rat thyroid C-cells. J. Histochem. Cytochem. 42(8), 1055–1060.

Throsby, M.; Lee, D.; Huang, W.; Yang, Z.; Copolov,D. L.; Lim, A. T. (1991) Evidence for atrial natriureticpeptide-(5-28) production by macrophages of therat spleen: An immunochemical and nonradioactiveinsituhybridizationapproach. Endo. 129, 991–1000.Throsby, M.; Yang, Z.; Lee, D.; Huang, W.; Copolov,D. L.; Lim, A. T. (1993) In vitro evidence for atrialnatriuretic factor-(5-28)productionby macrophagesof adult rat thymi. Endocrinol. 132 (5), 2184–2190.Tokushige, E.; Ito, K.; Ushikai, M.; Katahira, S.;Fukuda, K. (1994) Localization of IL-1" mRNA and cell adhesion molecules in the maxillary sinusmucosa of patients with chronic sinusitis. Laryngoscope 104, 1245–1250.Toran-Allerand, C. D.; Miranda, R. C.; Hochberg, R. B.; MacLusky, N. J. (1992) Cellular variations inestrogen receptor mRNA translation in the develop-ing brain: evidence from combined [125I] estrogenautoradiography and non-isotopic in situ hybridi-zation histochemistry. Brain Res. 576, 25–41.Trembleau, A.; Roche, D.; Calas, A. (1993)Combination of non-radioactive and radioactive insituhybridizationwith immunohistochemistry: anewmethod allowing the simultaneous detection of twomRNAs and one antigen in the same brain tissuesection. J. Histochem. Cytochem. 41, 489–498.Trembleau, A.; Morales, M.; Bloom, F. E. (1994)Aggregation of vasopressin mRNA in a subset ofaxonal swellings of the median eminence andposterior pituitary: Light and electron microscopicevidence. J. Neurosci. 14 (1), 39–53.Ueyama, T.; Houtani, T.; Nakagawa, H.; Baba, K.;Ikeda, M.; Yamashita, T.; Sugimoto, T. (1994) Asubpopulation of olivocerebellar projection neuronsexpress neuropeptide Y. Brain Res. 634, 353–357.Wani, G.; Wani, A. A.; Ambrosio, S. (1992) In situhybridization of human kidney tissue reveals cell-type-specific expression of the O6-methylguanine-DNA methyltransferase gene. Carcinogenesis 13(3), 463–468.Wani, G.; Wani, A. A.; Ambrosio, S. (1993) Cell type-specific expression of the O6-alkylguanine-DNAalkyltransferase gene in normal human liver tissuesas revealed by in situ hybridization. Carcinogenesis14 (4), 737–741.Whitman, G. F.; Pantazis, C. G. (1991) Cellular locali-zation of müllerian inhibiting substance messengerribonucleic acid during human ovarian folliculardevelopment. Am. J. Obstet. Gynecol. 165,1881–1886.Woodroofe, M. N.; Cuzner, M. L. (1993) CytokinemRNA expression in inflammatory multiple sclerosislesions: detection by non-radioactive in situhybridization. Cytokine 5 (6), 583–588.Xerri, L.; Monges, G.; Guigou, V.; Parc, P.; Hassoun,J. (1992) Detection of gastrin mRNA by in situhybridization using radioactive- and digoxigenin-labeled probes: a comparative study. APMIS 100,949–953.

186

References

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1.3 RNA target

A. Oligonucleotide Probesb. 3'-TailingNegro, F.; Pacchioni, D.; Shimizu, Y.; Miller, R. H.;Bussolati, G.; Purcell, R. H.; Bonino, F. (1992)Detection of intrahepatic replication of hepatitis Cvirus RNA by in situ hybridization and comparisonwith histopathology. Proc. Natl. Acad. Sci. USA89,2247–2251.Azum-Gelade, M.-C.; Noaillac-Depeyre, J.;Caizergues-Ferrer, M.; Gas, N. (1994) Cell cycleredistribution of U3 snRNA and fibrillarin. J. CellSci. 107, 463–475.

B. RNA probesBashir, R.; McManus, B.; Cunningham, C.;Weisenburger, D.; Hochberg, F. (1994) Detection of Eber-1 RNA in primary brain lymphomas inimmunocompetent and immunocompromisedpatients. J. Neuro-Oncol. 20, 47–53.Di Giuseppe, J. A.; Wu, T.-C.; Corio, R. L. (1994)Analysis of epstein-barr virus-encoded small RNA 1expression in benign lymphoepithelial salivarygland lesions. Mod. Pathol. 7 (5), 555–559.Lima, M. I.; Fonseca, M. E. N.; Flores, R.; Kitajima,E. W. (1994) Detection of avocado sunblotch viroidin chloroplasts of avocado leaves by in situhybridization. Arch. Virol. 138, 385–390.Lin, N.-S.; Chen, C.-C.; Hsu, Y.-H. (1993) Post-embedding in situ hybridization for localization of viral nucleic acid in ultra-thin sections. J.Histochem. Cytochem. 41 (10), 1513–1519.

C. DNA probesd. PCRTanaka, Y.; Enomoto, N.; Kojima, S.; Tang, L.; Goto, M.; Marumo, F.; Sato, C. (1993) Detection of hepatitis C virus RNA in the liver by in situhybridization. Liver 13, 203–208.

1.2 rRNA target

A. RNA probeCary, S. C.; Warren, W.; Anderson, E.; Giovannoni,S. J. (1993) Identification and localization ofbacterial endosymbionts in hydrothermal vent taxawith symbiont-specific polymerase chain reactionamplification and in situ hybridization techniques.Mol. Marine Biol. Biotechn. 2 (1), 51–62.Fischer, D.; Weisenberger, D.; Scheer, U.: Assigningfunctions to nucleolar structures. ChomosomaFocus.

B. Oligonucleotide probesa. 3'-EndlabelingDixon, B. (1992) Targeting ribosomal RNA:Identifying microbes. BioTechn. 10, 124.Zarda, B.; Amann, R.; Wallner, G.; Schleifer, K.-H.(1991) Identification of single bacterial cells usingdigoxigenin-labeled rRNA-targeted oligonucleo-tides. J. Gen. Microbiol. 137, 2823–2830.

b. 3'-TailingAzum-Gélade, M.-C.; Noaillac-Depeyre, J.;Caizergues-Ferrer, M.; Gas, N. (1994) Cell cycleredistribution of U3 snRNA and fibrillarin. Presencein the cytoplasmic nucleolus remnant and in theprenucleolar bodies at telophase. J. Cell Sci. 107,463–475.Concha, I. I.; Urzua, U.; Yanez, A.; Schroeder, R.;Pessot, C.; Burzio, L. O. (1993) U1 and U2 snRNAare localized in the sperm nucleus. Experiment. CellRes. 204, 378–381.

c. Chemical synthesisGersdorf, H.; Pelz, K.; Göbel, U. B. (1993)Fluorescence in situ hybridization for directvisualization of Gram-negative anaerobes insubgingival plaque samples. FEMS Immunol. &Medical Microbiol. 6, 109–114.Roller, C.; Wagner, M.; Amann, R.; Ludwig, W.;Schleifer, K.-H. (1994) In situ probing of Gram-positive bacteria with high DNA G+C content using23S rRNA-targeted oligonucleotides. Microbiol. 140,2849–2858.Wallner, G.; Amann, R.; Beisker, W. (1993)Optimizing fluorescent in situ hybridization withrRNA-targeted oligonucleotide probes for flowcytometric identification of microorganisms. Cytom. 14, 136–143.Zarda, B.; Amann, R.; Wallner, G.; Schleifer, K.-H.(1991) Identification of single bacterial cells usingdigoxigenin-labeled rRNA-targetedoligonucleotides.J. Gen. Microbiol. 137, 2823–2830.

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Gentilomi, G.; Musiani, M.; Zerbini, M.; Gallinella, G.;Gibellini, D.; La Placa, M. (1989) A hybrido-immuno-cytochemical assay for the in situ detection ofcytomegalovirus DNA using digoxigenin-labeledprobes. J. Immunol. Methods 125, 177–183.Genitilomi, G.; Musiani, M.; Zerbini, M.; Gibellini, D.;Gallinella, G.; Venturoli, S. (1992) Double in situhybridization for detection of herpes simplex virusand cytomegalovirus DNA using non-radioactiveprobes. J. Histochem. Cytochem. 40 (3), 421–425.Gentilomi, G.; Zerbini, M.; Musiani, M.; Gallinella,G.; Gibellini, D.; Venturoli, S.; Re, M. C.; Pileri, S.;Finelli, C.; La Placa, M. (1993) In situ detection ofB19 DNA in bone marrow of immunodeficientpatients using a digoxigenin-labeled probe. Mol.Cell. Probes 7, 19–24.Han, K.-H.; Hollinger, F. B.; Noonan, C. A.; Yoffe, B.(1992) Simultaneous detection of HBV-specificantigens and DNA in paraffin-embedded liver tissueby immunohisto-chemistry and in situ hybridizationusing a digoxigenin-labeled probe. J. Virol. Meth.37, 89–98.Heiles, H. B. J.; Genersch, E.; Kessler, C.; Neumann,R.; Eggers, H. J. (1988) In situ hybridization withdigoxigenin-labeledDNA of human papillomaviruses(HPV 16/18) in HeLa and SiHa cells. BioTechn. 6,978–981.Heino, P.; Hukkanen, V.; Arstila, P. (1989) Detectionof human papilloma virus (HPV) DNA in genitalbiopsy specimens by in situ hybridization withdigoxigenin-labeled probes. J. Virol. Methods 26,331–338.Holm, R.; Karlsen, F.; Nesland, J. M. (1992) In situ hybridization with nonisotopic probes usingdifferent detection systems. Modern Pathol. 5 (3),315–319.Han, K. H.; Hollinger, F. B.; Noonan, C. A.; Solomon,H.; Klintmalm, G. B. G.; Genta, R. M.; Yoffe, B.(1993) Southern-blot analysis and simultaneous in situ detection of hepatitis B virus-associated DNA and antigens in patients with end-stage liverdisease. Hepatol. 18, 1032–1038.Jackwood, D. J.; Swayne, D. E.; Fisk, R. J. (1992)Detection of infectious bursal disease virusesusing in situ hybridization and non-radioactiveprobes. Avian Diseases 36, 154–157.Jacob, J.; Kassir, R.; Kelsoe, G. (1991) In situstudies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. I. The architectureand dynamics of responding cell populations. J. Exp. Med. 173, 1165–1175.

2. DNA target

A. DNA probesa. RPLBoland, N. I.; Gosden, R. G. (1994) Clonal analysisof chimaeric mouse ovaries using DNA in situhybridization. J. Reprod. Fertility 100, 203–210.Brown, D. W.; Welsh, R. M.; Like, A. A. (1993)Infection of peripancreatic lymph nodes but notislets precedes kilham rat virus-induced diabetes inBB/Wor rats. J. Virol. 67 (10), 5873–5878.Burnett, S.; Kiessling, U.; Pettersson, U. (1989) Lossof bovine papillomavirus DNA replication control ingrowth-arrested transformed cells. J. Virol. 63 (5),2215–2225.Cenacchi, G.; Musiani, M.; Gentilomi, G.; Righi, S.;Zerbini, M.; Chandler, J. G.; Scala, C.; La Plaga, M.;Martinelli, G. N. (1993) In situ hybridization at theultrastructural level: localization of cytomegalovirusDNA using digoxigenin labeled probes. J. Submicrosc. Cytol. Pathol. 25 (3), 341–345.Clark, J.; Moore, L.; Krasinskas, A.; Way, J.; Battey, J.; Tamkun, J.; Kahn, R. A. (1993) Selectiveamplification of additional members of the ADP-ribosylation factor (ARF) family: Cloningof additionalhuman and drosophila ARF-like genes. Proc. Natl.Acad. Sci. USA90, 8952–8956.Coates, P. J.; D’Ardenne, A. J.; Slavin, G.; Kingston,J. E.; Malpas, J. S. (1993) Detection of Epstein-Barrvirus in reed-sternberg cells of hodgkin’s diseasearising in children. Med. Pediatric Oncol. 21, 19–23.Delvenne, P.; Fontaine, M.-A.; Delvenne, C.;Nikkels, A.; Boniver, J. (1994) Detection of humanpapillomaviruses in paraffin-embedded biopsies of cervical intraepithelial lesions: analysis byimmunohistochemistry, in situ hybridization, andthe polymerase chain reaction. Modern Pathol. 7(1), 113–119.Delvenne, P.; Kaschten, B.; Deneufbourg, J. M.;Demanez, L.; Stevenaert, A.; Reznik, M.; Boniver, J.(1993) Detection of Epstein-Barr virus in a case ofundifferentiated nasopharyngeal carcinoma by in situ hybridization with digoxigenin-labeled PCR-generated probes. Virchows Archiv A. Pathol. Anat.423, 145–150.Fletcher, S.; Darragh, D.; Fan, Y.; Grounds, M. D.;Fischer, C. J.; Beilharz, M. W. (1993) Specificcloning of DNA fragments unique to the dog Y chromosome. GATA10 (3–4), 77–83.Furuta, Y.; Inuyama, Y.; Nagashima, K. (1989)Detection of human papillomavirus genome innasolaryngeal papillomas using digoxigenin labeledDNA probes. J. Otolaryngol. Japan. 92, 2055–2063.Furuta, Y.; Shinohara, T.; Sano, K.; Meguro, M.;Nagashima, K. (1990) In situ hybridization withdigoxigenin-labeled DNA probes for detection ofviral genomes. J. Clin. Pathol. 43, 806–809.

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Premoli-de-Percoco, G.; Galindo, I.; Ramirez, J. L.(1992) In situ hybridization with digoxigenin-labeledDNA probes for the detection of human papilloma-virus-induced focal epithelial hyperplasia amongVenezuelans. Virchows Archiv A. Pathol. Anat. 420,295–300.Schmidt, P.; Meyer, H.; Hübert, P.; Hafner, A.; Andiel, E.; Grabner, A.; Dahme, E. (1994) Insituhybridization for demonstrationof equine herpes-virus type 1 DNA in paraffin wax-embedded tissuesand its use in horses with disseminated necrotizingmyeloencephalitis. J. Comp. Path. 110, 215–225.Schwarz, T. F.; Nerlich, A.; Hottenträger, B.; Jäger,G.; Wiest, I.; Kantimm, S.; Roggendorf, H.; Schultz,M.; Gloning, K.-P.; Schramm, T.; Holzgreve, W.;Roggendorf, M. (1991) Parvovirus B19 infection of the fetus. Histology and in situ hybridization. Am. J. Clin. Pathol. 96 (1), 121–126.Schwarz, T. F.; Nerlich, A.; Hillemanns, P. (1993)Detection of parvovirus B19 in fetal autopsies. Arch.Gynecol. Obstet. 253, 207–213.Smith, K. L.; Robbins, P. D.; Dawkins, H. J. S.;Papadimitriou, J. M.; Redmond, S. L.; Carrello, S.;Harvey, J. M.; Sterrett, G. F. (1994) c-erbB-2 amplification in breast cancer: Detection in formalin-fixed, paraffin-embedded tissue by in situhybridization. Hum. Pathol. 25, 413–418.Thompson, C. H.; Biggs, I. M.; De Zwart-Steffe, R. T.(1990) Detection of molluscum contagiosum virusDNA by in situ hybridization. Pathol. 22, 181–186.

b. Nick translationCoates, P. J.; Mak, W. P.; Slavin, G.; D’Ardenne, A. J.(1991) Detection of single copies of Epstein-Barrvirus in paraffin wax sections by non-radioactive in situ hybridization. J. Clin. Pathol. 44, 487–491.Coates, P. J.; Slavin, G.; D’Ardenne, A. J. (1991)Persistence of epstein-barr virus in reed-sternbergcells throughout the course of hodgkin’s disease. J. Pathol. 164, 291–297.Cooper, K.; Herrington, C. S.; Graham, A. K.; Evans, M. F.; McGee, J. O’D (1991) In situ humanpapillomavirus (HPV) genotyping of cervical intra-epithelial neoplasia in South African and British patients: Evidence for putative HPV integration in vivo. J. Clin. Pathol. 44, 400–405.Cooper, K.; Herrington, C. S.; Lo, E. S.-F.; Evans, M. F.; McGee, J. O. (1992) Integration of human papillomavirus types 16 and 18 in cervical adenocarcinoma. J. Clin. Pathol. 45, 382–384.Del Buono, R.; Fleming, K. A.; Morey, A. L.; Hall, P. A.; Wright, N. A. (1992) A nude mouse xenograft model of fetal intestine development and differentiation. Development 114, 67–73.Egawa, K.; Shibasaki, Y.; De Villiers, E.-M. (1993)Double infection with human papillomavirus 1 andhuman papillomavirus 63 in single cells of a lesiondisplayingonlyanhumanpapillomavirus 63-inducedcytopathogenic effect. Lab. Invest. 69 (5), 583–588.

Kawase, M.; Honda, M.; Niimura, M. (1994)Detection of human papillomavirus type 60 inplantar cysts and verruca plantaris by the in situhybridization method using digoxigenin labeledprobes. J. Dermatol. 21, 709–715.Keighren, M.; West, J. D. (1993) Analysis of cellploidy in histological sections of mouse tissues byDNA-DNA in situ hybridization with digoxigenin-labeled probes. Histochem. J. 25, 30–44.Konkle, B. A.; Shapiro, S. S.; Asch, A. S.; Nachman,R. L. (1990) Cytokine-enhanced expression ofglycoprotein Iba in human endothelium. J. Biol.Chem. 265 (32), 19833–19838.Lassus, J.; Niemi, K.-M.; Marjamäki, A.; Syrjänen, S.;Kartamaa, M.; Lehmus, A.; Krohn, K.; Ranki, A.(1992) Comparison of four in situ hybridizationmethods, based on digoxigenin- and biotin-labeledprobes, in detecting HPV DNA in male condylomataacuminata. Intern. J. STD & AIDS3, 196–203.Lau, J. Y. N.; Naoumov, N. V.; Alexander, G. J. M.;Williams, R. (1991) Rapid detection of hepatitis Bvirus DNA in liver tissue by in situ hybridization and its combination with immunohistochemistry for simultaneous detection of HBV antigens. J. Clin. Pathol. 44, 905–908.Lereuz, M.; Pallier, C.; Vassias, I.; Elouet, J. F.;Romeo, P.; Morinet, F. (1994) Differential transcription, without replication, of non-structuraland structural genes of human parvovirus B19 inthe UT7/EPO cell line as demonstrated by in situhybridization. J. Gen. Virol. 75, 1475–1478.Morey, A. L.; Ferguson, D. J. P.; Leslie, K. O.;Taatjes, D. J.; Fleming, K. A. (1993) Intracellularlocalization of parvovirus B19 nucleic acid at theultrastructural level by in situ hybridization withdigoxigenin-labeled probes. Histochem. J. 25,421–429.Morris, R. G.; Arends, M. J.; Bishop, P. E.; Sizer, K.;Duvall, E.; Bird, C. C. (1990) Sensitivity of digoxi-genin- and biotin-labeled probes of detection ofhuman papillomavirus by in situ hybridization. J. Clin. Pathol. 43, 800–805.Musiani, M.; Gentilomi, G.; Zerbini, M.; Gibellini, D.;Gallinella, G.; Pileri, S.; Baglioni, P.; La Placa, M.(1990) In situ detection of cytomegalovirus DNA in biopsies of AIDS patients using a hybrido-immunocytochemical assay. Histochem. 94, 21–25.Prelle, A.; Fagiolari, G.; Checcarelli, N.; Moggio, M.;Battistel, A.; Comi, G. P.; Bazzi, P.; Bordoni, A.;Zeviani, M.; Scarlato, G. (1994) Mitochondrialmyopathy: correlation between oxidative defect and mitochondrial DNA deletions at single fiberlevel. Acta Neuropathol. 87, 371–376.Permeen, A. M. Y.; Sam, C. K.; Pathmanathan, R.;Prasad, U.; Wolf, H. (1990) Detection of Epstein BarrVirus DNA in nasopharyngeal carcinoma using anon-radioactive digoxigenin-labeled probe. J. Virol.Meth. 27 (3), 261–268.

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Lawrence, J. B.; Marselle, L. M.; Byron, K. S.;Johnson, C. V.; Sullivan, J. L.; Singer, R. H. (1990)Subcellular localization of low-abundance humanimmunodeficiency virus nucleic acid sequencesvisualized by fluorescence in situ hybridization. Proc. Natl. Acad. Sci. 87, 5420–5424.Leitch, I. J.; Leitch, A. R.; Heslop-Harrison, J. S.(1991) Physical mapping of plant DNA sequences by simultaneous in situ hybridization of twodifferently labeled fluorescent probes. Genome 34,329–333.Morey, A. L.; Porter, H. J.; Keeling, J. W.; Fleming,K. A. (1992) Non-isotopic in situ hybridization andimmunophenotyping of infected cells in theinvestigation of human fetal parvovirus infection. J. Clin. Pathol. 45, 673–678.Scherthan, H. (1991) Characterisation of a tandemrepetitive sequence cloned from the deer capreoluscapreolus and it’s chromosomal localisation in twomuntjac species. Hereditas (in press).Steilen, H.; Ketter, R.; Romanakis, K.; Zwergel, T.;Unteregger, G.; Bonkhoff, H.; Seitz, G.; Ziegler, M.;Zand, K. D.; Wullich, B. (1994) DNA aneuploidy inprostatic adenocarcinoma: A frequent event asshown by fluroescence in situ DNA hybridization.Hum. Pathol. 25, 1306–1313.Troncone, G.; Herrington, C. S.; Cooper, K.; De Angelis, M. L.; McGee, J. O. (1992) Detection ofhuman papillomavirus in matched cervical smearsand biopsy specimens by non-isotopic in situhybridization. J. Clin. Pathol. 45, 308–313.Wachtler, F.; Schöfer, C.; Schedle, A.; Schwarzacher,H. G.; Hartung, M.; Stahl, A.; Gonzales, I.; Sylvester, J.(1991) Transcribed and nontranscribed parts of thehuman ribosomal gene repeat show a similarpattern of distribution in nucleoli. Cytogenet. CellGenet. 57, 175–178.Wood, N. B.; Sheikholeslami, M.; Pool, M.; Coon, J. S.(1994) PCR production of a digoxigenin-labeledprobe for the detection of human cytomegalovirusin tissue sections. Diagn. Mol. Pathol. 3 (3),200–208.

d. PCRSaeki, K.; Mishima, K.; Horiuchi, K.; Hirota, S.;Nomura, S.; Kitamura, Y.; Aozasa, K. (1993)Detection of low copy numbers of epstein-barr virus by in situ hybridization using nonradioisotopicprobes prepared by the polymerase chain reaction.Diagn. Mol. Pathol. 2 (2), 108–115.Varma, V. A.; Cerjan, C. M.; Abbott, K. L.; Hunter, S. B.(1994) Non-isotopic in situ hybridization method formitochondria in oncocytes. J. Histochem.Cytochem. 42 (2), 273–276.

Hegele, R. G.; Bicknell, S. G.; Bailey, D. J.; Cameron,R. G. (1994) In situ hybridization for the Y chromo-some reveals a donor origin for a posttransplantlymphoproliferative disorder in a sex-mismatchedhepatic allograft. Arch. Pathol. Lab. Med. 118,795–796.Heiskanen, M.; Karhu, R.; Hellsten, E.; Peltonen, L.;Kallioniemi, O. P.; Palotie, A. (1994) High resolutionmapping using fluorescence in situ hybridization toextended DNA fibers prepared from agarose-embedded cells. BioTechn. 17 (5), 928–933.Herrington, C. S.; Burns, J.; Graham, A. K.; Bhatt, B.; McGee, J. O. (1989) Interphase cyto-genetics using biotin and digoxigenin labeledprobes. II. Simultaneous differential detection ofhuman and papilloma virus nucleic acids inindividual nuclei. J. Clin. Pathol. (Lond) 42 (6),601–606.Herrington, C. S.; Burns, J.; Graham, A. K.; Evans,M.; McGee, J. O. (1989) Interphase cytogeneticsusing biotin and digoxigenin labeled probes. I.Relative sensitivity of both reporter molecules fordetection of HPV16 in caski cells. J. Clin. Pathol.(Lond) 42 (6), 592–600.Herrington, C. S.; Bhatt, B.; Burns, J.; Graham, A. K.;McGee, J. O. (1989) Simultaneous non-isotopic insitu hybridization using biotinylated and digoxigenin-labeled probes. J. Pathol. (Edin) 157, 163.Herrington, C. S.; De Angelis, M.; Evans, M. F.;Troncone, G.; McGee, J. O. (1992) Detection of highrisk human papillomavirus in routine cervicalsmears: Strategy for screening. J. Clin. Pathol. 45,385–390.Herrington, C. S.; Graham, A. K.; Burns, J.; McGee, J. O. (1989) Enhancement of sensitivity ofdigoxigenin labeled probes: the evaluation of a biotin independent detection system. J. Pathol.(Edin) 158, 337.Herrington, C. S.; Graham, A. K; McGee, J. O. (1991) Interphase cytogenetics using biotin and digoxigenin labeled probes: III. Increased sensitivityand flexibility for detecting HPV in cervical biopsyspecimens and cell lines. J. Clin. Pathol. 44 (1),33–38.Kerstens, H. M. J.; Poddighe, P. J.; Hanselaar, A. G. J. M. (1995) A novel in situ hybridization signal amplification method based on the depositionof biotinylated tyramine. J. Histochem. Cytochem.43 (4), 347–352.Kerstens, H. M. J.; Poddighe, P. J.; Hanselaar, A. G.J. M. (1994) Double-target in situ hybridization inbrightfield microscopy. J. Histochem. Cytochem. 42(8), 1071–1077.

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C. Oligonucleotide probeb. 3'-TailingCubie, H. A.; Felix, D. H.; Southam, J. C.; Wray, D.(1991) Application of molecular techniques in therapid diagnosis of EBV-associated oral hairyleukoplakia. J. Oral. Pathol. Med. 20, 271–274.Cubie, H. A.; Inglis, J. M.; McGowan, A. M. (1991)Detection of respiratory syncytial virus antigen andnucleic acid in clinical specimens using syntheticoligonucleotides. J. Virol. Meth. 34, 27–35.Oraveerakul, K.; Choi, C. S.; Molitor, T. W. (1993)Tissue tropisms of porcine parvovirus in swine.Arch. Virol. 130, 377–389.Pacchioni, D.; Papotti, M.; Bonio, F.; Bussolati, G.;Negro, F. (1992) Detection of cytomegalovirus by insituhybridizationusingadigoxigenin-tailed oligo-nucleotide. Liver 12, 257–261.

c. Chemical synthesisZischler, H.; Nanda, J.; Schäfer, R.; Schmid, M.;Epplen, J. (1989) Digoxigenated oligonucleotideprobes specific for simple repeats in DNAfingerprinting and hybridization in situ. Hum. Genet.82 (3), 227–233.

B. RNA probesBrown, C. C.; Meyer, R. F.; Grubman, M. J. (1994)Identification of african horse sickness virus in cellculture using a digoxigenin-labeled RNA probe. J. Vet. Diagn. Invest. 6, 153–155.Coen, E. S.; Romero, J. M.; Doyle, S.; Elliott, R.;Murphy, G.; Carpenter, R. (1990) Floricaula: A homeotic gene required for flower development in Antirrhinum majus. Cell 63, 1311–1322.Marquardt, D. L.; Walker, L. L.; Heinemann, S.(1994) Cloning of two adenosine receptor subtypesfrom mouse bone marrow-derived mast cells. J. Immunol. 152, 4508–4515.Su, I.-J.; Chen, R.-L.; Lin, D.-T.; Lin, K.-S.; Chen, C.-C. (1994) Epstein-Barr virus (EBV) infectsT lymphocytes in childhood EBV-associatedhemophagocytic syndrome in Taiwan. Am. J. Pathol.144 (6), 1219–1225.

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Celeda, D.; Aldinger, K.; Haar, F.-M.; Hausmann, M.;Durm, M.; Ludwig, H.; Cremer, C. (1994) Rapidfluorescence in situ hybridization with repetitiveDNA probes: Quantification by digital imageanalysis. Cytometry 17, 13–25.Chevillard, C.; Le Paslier, D.; Passage, E.; Ougen, P.;Billault, A.; Boyer, S.; Mazan, S.; Bachellerie, J. P.; Vignal, A.; Cohen, D.; Fontes, M. (1993)Relationship between Charcot-Marie-Tooth 1A andSmith-Magenis regions. snU3 may be a candidategene for the Smith-Magenis syndrome. Hum. Mol.Gen. 2 (8), 1235–1243.Christmann, A.; Lagoda, P. J. L.; Zang, K. D. (1991)Non-radioactive in situ hybridization pattern of theM13 minisatellite sequences on human metaphasechromosomes. Hum. Genet. 86, 487–490.Couturier, J.; Garau-Sastre, X.; Schneider-Maunoury, S.; Labib, A.; Orth, G. (1991) Integrationof papillomavirus DNA near myc genes in genitalcarcinomas and its consequences for proto-oncogene expression. J. Virol. Vol. 65, No. 8,4534–4538.Dal Cin, P.; Aly, M. S.; Delabie, J.; Ceuppens, J. L.;Van Gool, S.; Van Damme, B.; Baert, L.; Van Poppel,H.; Van Den Berghe, H. (1992) Trisomy 7 andtrisomy 10 characterize subpopulations of tumor-infiltrating lymphocytes in kidney tumors and in thesurrounding kidney tissue. Proc. Natl. Acad. Sci.USA89, 9744–9748.Dauwerse, J. G.; Wiegant, J.; Raap, A. K.; Breuning,M. H.; Ommen, van, G. J. B. (1992) Multiple colorsby fluorescence in situ hybridization using ratio-labeled DNA probes create a molecular karyotype.Hum. Mol. Gen. 1 (8), 593–598.Davies, A. F.; Barber, L.; Murer-Orlando, M.;Bobrow, M.; Adinolfi, M. (1994) FISH detection oftrisomy 21 in interphase by the simultaneous use of two differentially labeled cosmid contigs. J. Med.Genet. 31, 679–685.De Frutos, R.; Kimura, K.; Peterson, K. R. (1990) In situ hybridization of Drosophila polytenechromosomes with digoxigenin-dUTP labeledprobes. Methods Mol. Cell. Biol. 2, 32–36.Delhanty, J. D. A.; Griffin, D. K.; Handyside, A. H.;Harper, J.; Atkinson, G. H. G.; Pieters, M. H. E. C.;Winston, R. M. L. (1993) Detection of aneuploidyand chromosomal mosaicism in human embryosduring preimplantation sex determination byfluorescent in situ hybridzation (FISH). Hum. Mol.Gen. 2 (8), 1183–1185.De Leeuw, B.; Suijkerbuijk, R. F.; Balemans, M.;Sinke, R. J.; De Jong, B.; Molenaar, W. M.; Meloni,A. M.; Sandberg, A. A.; Geraghty, M.; Hofker, M.;Ropers, H. H.; Geurts van Kessel, A. (1993)Sublocalization of the synovial sarcoma-associatedt(X;18) chromosomal breakpoint in Xp11.2 usingcosmid cloning and fluorescence in situhybridization. Oncogene 8, 1457–1463.

Antonacci, R.; Marzella, R.; Finelli, P.; Lonoce, A.;Forabosco, A.; Archidiacono, N.; Rocchi, M. (1995) A panel of subchromosomal painting librariesrepresenting over 300 regions of the humangenome. Cytogenet. Cell Genet. 68, 25–32.Arnold, N.; Seibl, R.; Kessler, C.; Wienberg, J.(1992) Non-radioactive in situ hybridization withdigoxigenin labeled DNA probes. BiotechnicHistochem. 67 (2), 59–67.Arnoldus, E. P. J.; Wiegant, J.; Noordermeer, I. A.;Wessels, J. W.; Beerstock, G. C.; Grosveld, G. C.;van der Ploeg, M.; Raap, A. K. (1990) Detection ofthe Philadelphia chromosome in interphase nuclei.Cytogenet. Cell Genet. 54, 108–111.Ashley, T.; Ried, T.; Ward, D. C. (1994) Detection of nondisjunction and recombination in meiotic andpostmeiotic cells from XYSxr [XY, Tp(Y)1Ct] miceusing multicolor fluorescence in situ hybridization.Proc. Natl. Acad. Sci. USA91, 524–528.Avarello, R.; Pedicini, A.; Caiulo, A.; Zuffardi, O.;Fraccaro, M. (1992) Evidence for an ancestralalphoid domain on the long arm of humanchromosome 2. Hum. Genet 89, 247–249.Balazs, M.; Mayall, B. H.; Waldmann, F. M. (1991)Simultaneous analysis of chromosomal aneusomyand 5-bromodeoxyuridine incorporation in MCF-7breast tumor cell line. Cancer Genet. Cytogenet. 57,93–102.Boggs, B. A.; Chinault, C. (1994) Analysis ofreplication timing properties of human X-chromo-somal loci by fluorescence in situ hybridization.Proc. Natl. Acad. Sci. USA91, 6083–6087.Boschman, G. A.; Buys, C. H. C. M.; Van der Veen,A. Y.; Rens, W.; Osinga, J.; Slater, R. M.; Aten, J. A.(1993) Identification of a tumor marker chromo-some by flow sorting, DNA amplification in vitro,and in situ hybridization of the amplified product.Genes Chrom. Cancer 6, 10–16.Boyle, A. L.; Feltquite, D. M.; Dracopoli, N. C.;Housman, D. E.; Ward, D. C. (1992) Rapid physicalmapping of cloned DNA on banded mouse chromo-somes by fluorescence in situ hybridization.Genomics 12, 106–115.Brandt, C. A.; Hindkjaer, J.; Stromkjaer, H.;Pedersen, S.; Sunde, L.; Kolvraa, S. (1993)Molecular cytogenetics: Applications in clinicalgenetics. Europ. J. Obstetrics & Gynecol. Reprod.Biol. 50, 235–242.Brandt, C. A.; Lyngbye, T.; Pedersen, S.; Bolund, L.;Friedrich, U. (1994) Value of chromosome paintingin determining the chromosomal outcome inoffspring of a 12; 16 translocation carrier. J. Med.Genet. 31, 234–237.Celeda, D.; Bettag, U.; Cremer, C. (1992) PCR Amplification and simultaneous digoxigenin incorporation of long DNA probes for fluorescencein situ hybridization. BioTechn. 12 (1), 98–102.

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I NDEXCONTENTS

3. Chromosomes/Interphase nuclei

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Abbo, S.; Dunford, R. P.; Miller, T. E.; Reader, S. M.; King, I. P. (1993) Primer-mediated in situdetection of the B-hordein gene cluster on barleychromosome 1H. Proc. Natl. Acad. Sci. USA90,11821–11824.Brandt, C. A.; Djernes, B.; Stromkjaer, H.; Petersen,M. B.; Pedersen, S.; Hindkjaer, J.; Brinch-Iversen,J.; Bruun-Petersen, G. (1994) Pseudododicentricchromosome 18 diagnosed by chromosome paint-ing and primed in situ labeling (PRINS). J. Med.Genet. 31, 99–102.Brandt, C. A.; Kierkegaard, O.; Hindkjaer, J.; Jensen,P. K. A.; Pedersen, S.; Therkelsen, A. J. (1993) Ringchromosome 20 with loss of telomeric sequencesdetected by multicolour PRINS. Clin. Genet. 44,26–31.Cinti, C.; Santi, S.; Maraldi, N. M. (1993) Localiza-tion of single copy gene by PRINS technique. Nucl.Acids Res. 21 (24), 5799–5800.Gosden, J.; Hanratty, D. (1991) Comparison ofsensitivity of three haptens in the PRINS (oligo-nucleotide primed in situ DNA synthesis) reaction.Techniques 3 (4), 159–165.

Gosden, J.; Lawson, D. (1995) Instant PRINS: a rapid method for chromosome identification bydetecting repeated sequences in situ. Cytogenet.Cell Genet. 68, 57–60.Hindkjaer, J.; Koch, J.; Mogensen, J.; Pedersen, S.;Fischer, H.; Nygaard, M.; Junker, S.; Gregersen, N.;Kolvraa, S.; Therkelsen, A. J.; Bolund, L. (1991)Primed in situ labeling of nucleic acids. BFE8, (12)752–756.Hindkjaer, J.; Koch, J.; Terkelsen, C.; Brandt, C. A.;Kolvraa, S.; Bolund, L. (1994) Fast, sensitivemulticolor detection of nucleic acids in situ byPRimed IN Situ labeling (PRINS). Cytogenet. Cell Genet. 66,152–154.Hindkjaer, J.; Koch, J.; Mogensen, J.; Kolvraa, S.;Bolund, L. (1994) Primed in situ (PRINS) labeling ofDNA. Meth. Mol. Biol. 33, 95–109.

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4. Primed in SituLabeling of Nucleic Acids

5. In situhybridization in suspension

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Patel, V. G.; Shum-Siu, A.; Heniford, B. W.; Wieman,T. J.; Hendler, F. J. (1994) Detection of epidermalgrowth factor receptor mRNA in tissue sectionsfrom biopsy specimens using in situ polymerasechain reaction. Am. J. Pathol. 144 (1), 7–14.Pellestor, F.; Girardet, A.; Lefort, G.; Andréo, B.;Charlieu, J. P. (1994) Détection rapide deschromosomes 21 in situ par la technqie PRINS. Ann. Génét. 37 (2), 66–71.Pestaner, J. P.; Bibbo, M.; Bobroski, L.; Seshamma,T.; Bagasra, O. (1994) Potential of the in situpolymerase chain reaction in diagnostic cytology.Acta Cytol. 38, 676–680.Teyssier, M.; Carosella, E.; Gluckman, E.;Kirszenbau, M. (1994) PCR introcellulaire: nouvelleapproche de diagnostic tissulaire et cytogenetique.Immunoanal. Biol. Spec. 9, 159–164.Tsongalis, G. J.; McPhail, A. H.; Lodge-Rigal, R. D.;Chapman, J. F.; Silverman, L. M. (1994) Localized in situ amplification (LISA): A novel approach to in situ PCR. Clin. Chem. 40 (3), 381–384.Zehbe, I.; Hacker, G. W.; Rylander, E.; Sällström, J.;Wilander, E. (1992) Detection of single HPV copiesin SiHa cells by in situ polymerase chain reaction (in situ PCR) combined with immuno-peroxidaseand immunogold-silver staining (IGSS) techniques.Anticanc. Res. 12, 2165–2168.

6. PCR in situ technique

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General Introduction to In Situ Hybridization

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