Protocol Optimization for In Situ Hybridization and the Localization of Kiss2 Neurons in the Brain of

Polypterus senegalus

Tiffany Pang, UTRIP 2011

Introduction

Kisspeptin neurons are key players in the regulation of the hypothalamic pituitary gonadal (HPG)

axis and are important in the regulation of sexual behaviors and reproduction in animals. In the HPG axis,

the GnRH neurons release gonadotropin-releasing hormone to the pituitary, which releases luteinizing

hormone (LH) and follicle stimulating hormones (FSH) to the gonads, which would then release either

estradiol or testosterone downstream to effect some changes in reproductive or sexual behavior. The

estradiol or testosterone would then feed back to the hypothalamus to either release more and less

neurons (Akazome, Kanda, Okubo, & Oka, 2010; Kandel, Schwartz, & Jessell, 2000). One of the prevailing

mysteries is the so-called “missing link” to describe how steroid feedback occurs when GnRH neurons lack

estrogen receptor alpha that is essential for reproduction.. Kisspeptin neurons, which do possess estrogen

receptor alpha, are now the candidates to be one of the most important regulators of reproduction in

mammals.

There are currently two paralogous genes for kisspeptin: kiss1 and kiss2 genes. Only Kiss1 was

identified and has been the focus in studies on mammalian species because most mammals except for

monotremes lack Kiss2. However, recent studies have shown the existence of both kiss1 and kiss2 in wide

variety of species in vertebrates, thus understanding of both paralogs is necessary for the understanding of

kisspeptin systems in vertebrate in general. It is reported that some of the kiss1 or kiss2 neurons show

significant steroid sensitivity, which characterize kisspeptin neurons. However, the kisspeptin neurons that

show steroid sensitivity in each species varies as kiss1 or kiss2 neurons, which makes it difficult to

understand the functions of kisspeptin systems in vertebrates. To understand the significance of their loss

and subtypes to be expression in each nucleus that shows steroid sensitivity, we must study their

localization and determine the function of kiss1 and kiss2 as they evolved.

Some studies examined the localization and steroid sensitivity of kiss1 and kiss2 neurons in teleosts.

However, it was difficult to determine which population corresponds to what function in mammalian

species because whole genome duplication events accelerate the gene evolution, and may caused drastic

changes in morphology and expression patterns in teleosts. Here, to make the bridge over the

understandings between tetrapods and teleosts, intermediate species were studied. Third round whole

genome duplication (3R) of teleosts accelerated the gene evolution speed so much that it complicated the

connection between teleosts and tetrapods. Here, because the Polypterus senegalus is a ray-finned fish but

not a teleost, in other words they were diverged from teleosts just prior to 3R whole genome duplication

event, making it the perfect control for genome evolution studies on kiss1 and kiss2 (Mulley & Holland,

2004). Because the loss of kiss1 is suggested, the present study examined the localization of kiss2 in P.

senegalus using in-situ hybridization and compares its localization with corresponding brain structures in

Oryzias latipes, a ray-finned teleost fish whose speciation was after the whole genome duplication.

Materials and Methods

Cryosectioning

P. senegalus were maintained in aquaria (27°C; 14h light, 10h dark). Brain tissue was extracted

from the P. senegalus following cold anesthetization and fixed overnight in 4% paraformaldehyde/PBS. To

cryoprotect the tissue, the brains were submerged in 30% sucrose/PBS the next day for 4 hours or until

they sank to the bottom of the tube. Brain tissue was embedded in an agarose-based gel (5% low-melting

temperature agarose Sigma Type IX A and 20% sucrose). The compound was then frozen with a 10-second

exposure to -80°C n-hexane and mounted onto a specimen disc with OCT compound (Sakura finetek). 30

μm coronal brain sections were obtained using a Leica CM3050 S cryostat (OT = -24°C; CT = -28°C).

Sections were stored at -80°C until they were used.

Preparation of sense and anti-sense Kiss2 RNA probes

Kiss2 cDNA fragment was cloned by Akazome-sensei. The DNA fragment was amplified by PCR for

gene specific sense and anti-sense primers. For anti-sense transcription, polypkiss2 SE2 primers were used

while, for sense transcription, NUP primers were used. Both tubes were heated to 98°C for 1 minute and

following 30 cycles of 98°C for 10 seconds, 55°C for 15 seconds, 72°C for 1 minute. DNA fragments were

separated by 1.5% agarose gel electrophoresis and stained with Midori Green to visualize DNA by visual

light. The fragment was excised and eluted using a kit. 200-300 ng/kb DNA fragment was cloned into

pGEM-T plasmid (Promega). For labeling, a DIG labeling mixture was used during in vitro transcription.

Probes were assessed for their quality and purity by agarose gel electrophoresis and quantified by

spectrophotometry. The probes were store in -20°C until future use.

Protocol Optimization for P. senegalus Gender Determination

Hematoxylin and Eosin staining of P. senegalus gonadal tissue

Our initial protocol for hematoxylin and eosin staining consisted of the following 35 micrometer

cryosections prepared according to the aforementioned protocol were dried in a heater at 49°C for 30

minutes and then placed in 1x PBS for 1 minute. The slides were then dipped in a hematoxylin (Sigma) dye

bath for 3 minutes and washed under light running tap water for 1 hour. The slides were then dipped in an

Eosin (Wako) dye bath for 30 seconds and washed under light running tap water for 1 minute. The slides

were subsequently dehydrated and cover-slipped and visualized in a microscope.

Unfortunately, neither the hematoxylin nor the eosin dyes were taken up by the tissue adequately

and the dyed tissue had various colors not present in the dye baths. Because the protocol was written

originally for paraffin sections, hematoxylin and eosin staining must be optimized for cryosections. Using

recommendations from Protocol Online, the protocol was revised thusly:

The cryosections were heat-dried at 49°C for 1 hour. The gonadal tissue sections were then

dehydrated by immersing them in 70% alcohol for 30 seconds. They were then dipped into 95% and 100%

alcohol for 2 minutes, respectively. The slides were then placed in a hematoxylin (Sigma) blue dye bath for

5 minutes and then washed under running tap water in a Coplin jar for 10 minutes. The slides were

immersed again in 100% alcohol to dehydrate the sections for 2 minutes and then placed in an Eosin

(Wako) red dye bath for 30 seconds. The dye was washed off with copious amounts of tap water. The

gonadal tissue sections were then sequentially dehydrated in 70%, 95%, 100% (I and II), and Xylene (I and

II), cover-slipped, and visualized in bright-field microscopy.

Upon revision of the protocol for gonadal tissue cell visualization, both hematoxylin and eosin

stains were taken up by the tissue, and the cell nuclei and cytoplasm were clearly distinguished upon

bright-field microscopy (Figure 1). Upon comparison to images of hematoxylin and eosin staining of

goldfish ovaries, we concluded that female Polypterus ovaries were characterized by dense clusters of cell

nuclei while male Polypterus testes are characterized by fatty cells and sporadic distribution of cell nuclei

(Figure 1, 2). Moreover, on a macro scale, female gonadal tissues are black and characterized by rough,

small circular sacs while male gonadal tissues are yellow and have a smooth surface.

A B

Figure 1. Hematoxylin and Eosin staining of P. senegalus gonadal tissue using old and revised protocols. Both tissue

come from the same animal. Left. Using the protocol optimized for paraffin sections, unexpected miscellaneous

yellow and green colors appeared and staining was poor overall. Right. Using the protocol optimized for

cryosections, only the expected purple and violet colors were visualized. Cell cytoplasm and nuclei are clearly

stained; the darkened ovals are small sperm cells while the larger circles are fatty tissue that surround the male

gonads.

A B

Figure 2. Using the new protocol optimized for cryosections, female gonadal tissue cells were distinguished from

male gonadal tissue. Left. Female ovaries were characteristically large and round in morphology with dense

clusters of egg cells encased in sacs. Right. A close-up of the individual ovum encased in the larger membrane.

Protocol Optimization for localization of kiss2 in P. senegalus

Our initial protocol, optimized for performing in situ hybridization on O. latipes, yielded faint to no

staining. Originally, cryosections were incubated at 37°C for 3 hours. Following incubation, sections were

fixed in 4% paraformaldehyde/PBS and then washed twice in 1x PBS. Here, sections may undergo optional

proteinase K treatment. The sections then underwent acetylation of endogenous DNA by immersion in 0.25%

acetic anhydride in 0.1M Triethanolamine (TEA) solution. The sections were washed twice again in PBS

before adding 58°C hybridization buffer (HB) onto each slide for a 20 minute 58°C incubation period.

Afterwards, denatured kiss2 RNA probes (1 μL/mL HB) were added to the slides, and the slides were

incubated at 58C overnight. On the next day, sections were immersed in 50% formamide/2X SSC twice for

15 minutes at 58°C, TNE for 10 minutes at 37°C, TNE with RNase for 30 minutes at 37°C, TNE again for 10

minutes at 37°C, 2X SSC twice for 15 minutes at 58°C, 0.5X SSC twice for 15 minutes at 58°C, and then

washed with DIG-1 solution twice for 15 minutes. The sections were then blocked for immunological

detection with 1.5% blocking reagent (Roche) for 1 hour and then washed again with DIG-1. Anti-DIG

antibody (1:5000 DIG-1) was poured onto the blocked slides and rested for 1 hour. The sections were

washed again in DIG-1 and kept in DIG-1 for 15 minutes or overnight. Following the DIG-1 wash, the

sections were immersed in DIG-3 for 3 minutes. The sections were then taken out of the wash, and

detection solution was added and allowed to develop for a few hours or overnight. To end the reaction,

stopping reaction solution was then added. The sections were fixed in Bouin’s solution for 15 minutes,

serially dehydrated, and cover-slipped with paramount.

After a series of trials yielding faint signals or too much noise, three key factors that affected how

well the signals turned out were isolated: whether to digest proteins with ProK, whether to leave slides in

DIG-1 for 15 minutes or overnight, whether to develop slides for a few hours or overnight (Figure 3). A 2-

by-2-by-2 experiment using gnrh1 RNA probes was set up to determine how each factor attributed to the

quality of kiss2 signal:

Eight adjacent sections would be prepared on 4 slides were prepared from one P. senegalus. For example,

slides in the No TritonX-ProK0 would undergo neither Triton X nor proteinase K treatment, and on one

group of sections would be treated with 1 μL/mL of GnRH1 RNA probes while the other group would be

treated with 10 μL/mL probes. In this case, GnRH1 RNA probes, as opposed to kiss2 RNA probes, were

used because the location of GnRH neurons in P. senegalus has already been determined by

immunohistochemistry and, thus, can be used as a control experiment to determine their location by in-situ

hybridization. Following the protocol outlined above, using overnight immersion in DIG-1 and overnight

development, proteinase K treatment with no Triton X treatment yielded the best signals (Figure 4, G).

There did not seem to be any significant difference between probe concentrations. (For protocol for best

signals, please refer to the attached document.)

For the nomenclature of Polypterus brain nuclei, an atlas of the polypterus brain compiled by

Holmes and Northcutt (2003) was used.

Figure 3. Too little signal and too much noise were observed with small variations to the original protocol. Left.

With sections treated with neither ProK nor Triton X, using 1 μL/mL of GnRH1 RNA probe, and immersed in DIG-1 overnight, the signal was weak and imperceptible at low magnification. Right. When treated with ProK without

Triton X, using 1 μL/mL of GnRH RNA probe, and immersion in DIG-1 for 15 minutes, there was too much background staining.

A. ProK0/Yes Triton X – 1 μL /mL

B. ProK0/Yes Triton X – 10 μL/mL

C. ProK0/No Triton X – 1 μL /mL

D. ProK0/No Triton X – 10 μL /mL

E. ProK10/Yes Triton X – 1 μL/mL

F. ProK10/Yes Triton X – 10 μL/mL

G. ProK10/No Triton X - 1 μL/mL

H. ProK10/No Triton X – 10 μL/mL

Figure 4. GnRH1 in caudal olfactory epithelium. Above are 20 micrometer cryosections. In conditions A – D,

sections showed low to no signal. Small clusters of cells were clearly visible in the ventral area of the caudal

olfactory epithelium (E – H) and anterior olfactory bulb (not shown). The best signals were obtained using

condition G.

Results

Localization of GnRH1 and kiss2 neurons

Due to time constraints, localization of kiss2 neurons using the new optimized protocol could not

be performed. On the other hand, GnRH1 neurons were found to be localized in the ventral area of the

caudal olfactory epithelium and anterior olfactory bulb. Comparing with immunohistochemical data on the

localization of GnRH (Li, 2010), we have confirmed that GnRH1 exists in the anterior areas where Li had

found GnRH cell bodies and that GnRH cell bodies found in the posterior parts of the brain (i.e., midbrain,

preoptic area, hypothalamus, telencephalon, torus semicircularis, ventrolateral medulla) were most likely

GnRH2 cell bodies.

Figure 5. A comparison of non-specific GnRH cell body to GnRH1 localization by in-situ hybridization. GnRH1

neurons were found only where the blue line indicates, in the caudal olfactory epithelium and anterior olfactory

bulb, suggesting that the distinct GnRH clusters in the midbrain are GnRH2 neurons. Top image from Li, 2010.

Bottom image from Holmes & Northcutt, 2003.

Because of time constraint issues, we were able to localize kiss2 neurons using data obtained from

a P. senegalus that was processed using the original protocol. Though faint, some of the cell nuclei can be

observed at higher magnification. At least two distinct populations kiss2 DNA were found in the ventral

nucleus of area ventralis (Vv), postcommissural nucleus of area ventralis (Vp), and periventricular nucleus

of the posterior tuberculum (TPp) (Figure 6). It may be possible that the neuron populations in the ventral

nucleus of area ventralis and postcommissural nucleus of area ventralis are one and the same.

A B

C

Figure 6. Dispersed kiss2 cell bodies in the ventral nucleus of area ventralis (A), postcommissural nucleus of area

ventralis (B), and perventricular nucleus of the posterior tuberculum. All three sections are 40 micrometers thick

and were neither treated with ProK nor Triton X before reacting with gnrh1 RNA probes (1 uL/mL).

Discussion

Firstly, we examined optimization of the in-situ hybridization protocol of kiss2 mRNA for

polypterus fish, using a previous in-situ hybridization protocol optimized for medaka (O. latipes). The

primary differences were the mandatory application of proteinase K and overnight DIG-1 wash. Proteinase

K treatment of the sections significantly increased gnrh1 mRNA signals as proteinase K had rid the tissue of

nonspecific proteins while an overnight DIG-1 wash significantly reduced background noises by washing

away unbound anti-DIG antibodies. For medaka, proteinase K treatments and overnight DIG-1 washes are

optional. The differential treatment of polypterus tissue suggests that properties of medaka and polypterus

tissue are different in that polypterus tissue tends to retain anti-DIG antibodies, suggesting a better affinity

for the protein, and proteins in polypterus tissue tend to block signals, suggesting either there are more

proteins or a relatively smaller affinity for alkaline phosphatase on said tissue to bind with NBT and BCIP.

Secondly, with at least two distinct populations of kiss2 neurons, we can make meaningful comparisons to medaka. In medaka, kiss1 neurons are localized in the nucleus ventralis tuberis (NVT),

nucleus posterioris periventricularis (NPPv), and habenula, while kiss2 neurons are localized in the

nucleus recessus lateralis (NRL) (Kanda, et al., 2008) (Kitahashi, Ogawa, & Parhar, 2009). In P. senegalus,

kiss2 neurons seem to be localized in the Vv, Vp, and TPp. Though not completely equal structures, TPp in

polypterus is comparable to the NVT and NPPv regions in medaka, suggesting that there had been the kiss2

gene expression had changed its localization from anterior hypothalamic regions to the more caudal NRL.

Because it is known that the NVT in medaka is involved in steroid feedback via kiss1 neurons, we can

understand how the kiss1 and kiss2 neuron function changed. It would be interesting to compare whether

kiss2 neurons in the polypterus participate in steroid feedback the same way it does in medaka due to its

physical location in the brain or whether there is a compensation of function due to its change in location.

This bears particular resemblance to the differences between gnrh1 and gnrh2 neurons. Depending on its

area of expression and whether the species had gnrh1, gnrh2, and/or gnrh3, the neurons took on the

function of the other as a way of compensating for its loss or absence, which may what has happened for

kiss1 and kiss2 (Oka, 1997). As for kiss2 neurons in Vv and Vp, which are comparable to brain structures

that regulate homeostasis in teleost and sexual behaviors in goldfish, respectively, further in-situ

hybridization studies comparing kiss1 and kiss2 in polypterus would elucidate their function and purpose.

Moreover, to confirm the localization of kiss2 in polypterus, further in-situ hybridization should be

performed utilizing the new protocol optimized for the polypterus brain.

However, because it is unclear whether the three locations in which kiss2 were found in the

polypterus brain were all distinct kiss2 populations, ongoing in-situ hybridization using continuous

sections as opposed to adjacent sections would reveal the neurons more clearly. Use of larger adult

polypterus fish and the optimized protocol may also reveal another population of kiss2 neurons in the

preoptic area of the hypothalamus, where we had initially expected kiss2 expression to be.

In comparing the localization of kiss2 neurons among polypterus bichirs, medaka, and goldfish with

respect to the 3R whole genome duplication, current data suggests that, after 3R, the localization of kiss2

differed significantly. In the polypterus bichir, kiss2 neurons were localized in Vv, Vp, and TPp, which is

comparable to NVT in medaka. Of these, Vv and Vp are located in the telencephalic region while TPp is

located in the anterior optic tectum. In goldfish and medaka, kisspeptin neurons were found in areas more

similar to each other but not with polypterus bichirs: preoptic area, nucleus recessus lateralis (NRL),

habenula, NVT, nucleus lateralis tuberis (NLT); all of which are in the midbrain area. In goldfish, kiss2 is

expressed in three distinct populations: the preoptic area, NRT, and NRL; while kiss1 is expressed in the

habenula. In medaka, kiss2 is expressed in only one population in the NRL while kiss1 is expressed in NVT,

hypothalamic nuclei, and, like in goldfish, the habenula. The only steroid sensitive populations, however,

are kiss2 neurons in the preoptic area for goldfish and kiss1 neurons in NVT for medaka. Whereas the

localization varied significantly between polypterus and teleosts, variation of kiss2 localization within

teleosts seem to be a function of the kisspeptin system, in which one paralog of kisspeptin can compensate

for the absence of the other. The number of distinct populations and the general localization did not change

between teleosts, but between polypterus and teleosts, the only comparable area that was common among

all three fish to express any paralog of kisspeptin was TPp (polypterus)/preoptic area (goldfish)/NVT

(medaka). In goldfish and medaka, these areas are also the populations of kisspeptin neurons that are

steroid sensitive, suggesting that, prior to 3R whole genome duplication, kisspeptin populations in the

preoptic area may be directly involved in regulating reproduction.

Thirdly, data collected from the protocol optimization experiment has also elucidated the

localization of gnrh1 and gnrh2 neurons. Using Li’s immunohistochemical localization of gnrh in polypterus

(2010), we found with high certainty that gnrh1 neurons were localized in the olfactory epithelium and

olfactory bulb while gnrh2 were localized in the populations identified in the midbrain regions. This bears

particular importance because, as mentioned previously, gnrh1 and gnrh2 are sister genes to kiss1 and

kiss2 genes and share similar divergence. By comparing gnrh1 and gnrh2 neuron function in polypterus to

existing data on medaka gnrh1, gnrh2, and gnrh3 neuron function, in which gnrh2 and gnrh3 neurons are

neuromodulatory while gnrh1 neurons play a part in releasing GnRH (Oka, 2010), it would further

supplement the current study’s aim to explore the significance of 3R whole genome duplication.

Works Cited

Akazome, Y., Kanda, S., Okubo, K., & Oka, Y. (2010). Functional and evolutionary insights into vertebrate

kisspeptin systems from studies of fish brain. Journal of Fish Biology, 76, 161-182.

Holmes, P. H., & Northcutt, G. T. (2003). Connection of the Pallial Telencephalon in the Senegal Bichir,

Polypterus. Brain, Behav Evol, 61, 113-147.

Kanda, S., Akazome, Y., Matsunaga, T., Yamamoto, N., Yamada, S., Tsukamura, H., et al. (2008). Identification of

KiSS-1 product kisspeptin and steroid-sensitive seually dimorphic kisspeptin neurons in medaka (Oryzias

latipes). Endocrinology, 2467-2476.

Kandel, E., Schwartz, J., & Jessell, T. (2000). Principles of Neural Science. New York: McGraw-Hill.

Kitahashi, T., Ogawa, S., & Parhar, I. (2009). Cloning and expression of kiss2 in the zebrafish and medaka.

Endocrinology, 150, 821-831.

Li, L. (2010). Neurobiological analysis of Gonadotropin-releasing (GnRH) systems in the brain of the bichir,

Polypterus senegalus. Tokyo: UTRIP.

Mulley, J., & Holland, P. (2004). Comparative genomics: Small genome, big insights. Nature, 431, 916-917.

Oka, Y. (1997). The gonadotropin-releasing hormone (GnRH) neuronal system of fish brain as a model system for

the study of peptidergic neuromodulation. In I. Parhar, & Y. Sakuma, GnRH neurons: genes to behavior

(pp. 245-276). Tokyo: Brain Shuppan Publishers.

Oka, Y. (2010). Electrophysiological Characteristics of Gonadotrophin-Releasing Hormone1–3 Neurones: Insights

From a Study of Fish Brains. Journal of Neuroendrocrinology, 22, pp. 659-663.

Protocol for In-situ Hybridization in Polypterus Fish

DAY 1 - RECIPE

1. ProK10

Proteinase K (TaKaRa) 10 mL

1M Tris-HCL pH 8.0 20 mL

0.5M EDTA pH 8.0 20 mL

UPW 160 mL

2. Glycine PBS

Glycine 0.4 g

1X PBS 200 mL

3. 0.1M TEA

Triethanolamine 3.71 g

milliQ water 200 mL

10N NaOH 1.32 mL

Acetic anhydride 500 μL (add just before immersing slides in 0.1 M TEA)

DAY 1 – use RNA grade reagents

1. Incubate cryosections on MAS-GP slides at 37°C for 3 hours. 2. Make dike around sections with in-situ grade PAP pen to avoid leakage on the slide. 3. Post-fix with 4% PFA/PBS 5 minutes at room temperature 4. Wash with 1x PBS 5 minutes x 2 at room temperature 5. Treat with ProK 15 minutes 37°C water bath 6. Wash with 4% PFA/PBS 15 minutes at room temperature 7. Wash in Glycine PBS 10 minutes at room temperature 8. Wash in PBS 5 minutes at room temperature 9. Acetylate in 0.25% acetic anhydride in 0.1M TEA 10 minutes room temperature 10. Preparation for hybridization

10 mL stock formamide, 10 mL 1X PBS, 500 μL hybridization buffer in moisture chamber

11. Add 500 μL of 58°C HB buffer to each slide. 12. Place slides in moisture chamber and incubate at 58°C for at least 20 minutes. 13. Denature of probes

Heat HB buffer (500 μL/mL) in tubes 10 minutes 85°C

Mix DIG-labeled probes in HB buffer

Heat tubes 3 minutes 85°C

Cool tubes in ice and wait until tube heater temperature falls to 58°C

Heat tubes 15 minutes 58°C

12. Pour denatured probes onto slides (500 μL/mL) and incubate at 58°C overnight

DAY 2 - RECIPE

1. 50% formamide/2x SSC

Formamide (stock) 200 mL

20x SSC 40 mL

milliQ water 160 mL

2. 2X SSC

20X SSC 40 mL

UPW 360 mL

3. 0.5X SSC

20X SSC 10 mL

UPW 390 mL

4. TNE

1M Tris-HCl pH 7.6 2.5 ml

5M NaCl 25 ml

0.5M EDTA pH 8.0 0.5 ml

UPW 222 ml

5. TNE (1, 3)

TNE 200 mL

6. TNE (RNase)

TNE 200 mL

RNase 360 μL (add just before adding slides)

7. DIG-1

1M Tris-HCl pH 7.5 100 mL

5M NaCl 33.4 mL

milliQ water 866.6 mL

Tween20 1 mL

DAY 2 – use DNA grade reagents

1. Prepare two water baths at 37°C and 58°C. 2. Place slides in

50% formamide/2X SSC 15 minutes x 2 58°C

TNE (1) 15 minutes 37°C

TNE (RNase) 30 minutes 37°C

TNE (3) 15 minutes 37°C

2X SSC 15 minutes x 2 58°C

0.5X SSC 15 minutes x 2 58°C

3. Rinse in DIG-1 5 minutes at room temperature 4. Block for immunological detection with 1.5% Blocking Reagent

60 minutes at room temperature

5. Pour anti-DIG antibody (1:5,000 DIG-1) onto blocked slides 60 minutes at room temperature

6. Rinse in DIG-1 5 minutes x1 at room temperature Overnight at room temperature

DAY 3 - RECIPE

1. DIG-3

1M Tris-HCl pH 9.5 25 mL

5M NaCl 5 mL

1M MgCl2 12.5 mL

milliQ water 207.5 mL

2. Detection solution

DIG-3 10 mL

100 mg/mL NBT 33.75 μL

50 mg/mL BCIP 35 μL

DAY 3 – use DNA grade reagents

1. Wash in DIG-3 5 minutes at room temperature

2. Pour detection solution on slides Overnight at room temperature

DAY 4

1. Pour stopping reaction solution 5 minutes at room temperature

2. Fix in Bouin’s solution 15 minutes at room temperature

3. Serial dehydration in ethanol and xylene

70% ethanol 20 seconds

95% ethanol 20 seconds

100% ethanol (I) 20 seconds

100% ethanol (II) 20 seconds

Xylene (I) 1 minute

Xylene (II) 2 minute – 10 minutes

4. Cover-slipped with paramount.