Y Clin Pathol 1994;47:448-452

Microwave antigen retrieval inimmunocytochemistry: a study of 80 antibodies

E C Cuevas, A C Bateman, B S Wilkins, P A Johnson, J H Williams, A H S Lee,D B Jones, D H Wright

AbstractAins-To evaluate the effect ofmicrowave irradiation on the stainingquality of a range of commonly used pri-mary antibodies in archival, fornalinfixed, paraffin wax embedded material,with emphasis on antibodies that havepreviously worked successfully only onfrozen tissue.Methods-Immunocytochemistry (strep-tavidin-biotin complex technique) wasperformed on histological sections of arange ofnormal and pathological tissues,after varying treatment with microwaveirradiation. The staining quality of eachantibody was compared with thatachieved without prior treatment of thesections or after enzyme predigestion.Results-Microwave irradiation permit-ted successful unostaining with 20

Department of antibodies that stained only frozen tis-Histopathology, sues before. The staining characteristicsHospital, of 21 antibodies that were already knownSouthampton to stain formalin fixed, paraffin wax

ECCuevas embedded material were improved.A C Bateman Another 39 antibodies did not showB S Wilkins enhanced staining with microwave irra-P A Johnson diation. The method preserves tissueAH S Lee morphology and produces more consis-D B Jones tent staining than that achieved byD H Wright enzyme predigestion with many antibod-Correspondence to: ies. Microwave irradiation may also allowDr A C Bateman 4

Accepted for publication some primary antibodies to be used at25 November 1993 higher working dilutions. The citrate

Table 1 Monoclonal antibodies previously not reactive with formalin fixed, paraffin waxembedded tissue, which work well after microwave irradiation

Antibody CDlspecificity Source

Lymphocytic and myelomonocytic markersEll CD35 K Gatter, OxfordAT13/5 CD38 A Tutt, Tenovus, SouthamptonTU69 CD25 A Ziegler, BerlinWR16 CD45R Wessex Regional Immunology

UnitIB5 MHC class II ICRFIgD IgD DakoQBEND 10 CD34 NovocastraEBM 11 CD68 K Gatter, OxfordCD5 CD5 K Gatter, OxfordCD8 CD8 K Gatter, Oxford96TCS Human ybTCR Becton DickinsonBRIC 128 CD55 D Anstee, NBTS, Bristol"5Proliferation markersMIB 1 Ki-67 antigen J Gerdes, Forschungsinstitut

Borstel, Germany7JC1 Proliferation marker K Gatter, Oxford"6Ki-67 Ki-67 antigen J Gerdes, Forschungsinstitut

Borstel, Germany'7Adhesion molecules1-4C3 VCAM-1 D Haskard, HammersmithBRIC 235 CD44 D Anstee, NBTS, Bristol"2H1 CD44 S Jalkenen, Turku, FinlandIF1 CD44 S Jalkenen, Turku, FinlandIE8 CD44 S Jalkenen, Turku, Finland

buffer used in this study avoids the neces-sity of exposure to heavy metal salts.Conclsions-Microwave antigen retrievalrepresents an important technicaladvance within immunocytochemistrythat will greatly increase the range ofantibodies which can be used to studyformalin fixed, paraffin wax embeddedtissues.

( Clin Pathol 1994;47:448-452)

Microwave irradiation has been used toimprove tissue fixation,'-2 to enhance histolog-ical staining for light and electronmicroscopy,3-4and, more recently, to enhanceimmunocytochemical staining.5-'0 The lattertechnique is becoming increasingly popularand has several advantages over conventionalmethods such as enzymatic predigestion." Shiet al have documented a range of antibodies inwhich performance in fixed tissue sectionswas improved by microwave irradiation.6Many of the antibodies they investigated,however, were already known to be active infixed tissue. The purpose of the present studywas to extend the use of the technique specifi-cally to antibodies previously found to reactonly with frozen sections or cytological prepa-rations.

MethodsEighty antibodies were studied, consisting of47 reactive with lymphohistiocytic antigens,20 reactive with cell adhesion molecules, fourproliferation markers, and nine other antibod-ies directed against miscellaneous antigens.All except the CD3 reagent were monoclonalantibodies. A range of normnal tissues (lymphnode, tonsil, spleen, bone marrow, smallbowel) and tissues showing a variety ofpathologies (glomerulonephritis, Hashimoto'sthyroiditis, Hodgkin's and non-Hodgkin'slymphomas, cerebral astrocytomas, sarcoido-sis, Crohn's disease) were studied. All material,obtained from the files of the Department ofPathology at Southampton General Hospital,was fixed in 10% neutral buffered formalinand embedded in paraffin wax using routinemethods.

Tissue sections were cut at 5 Rm on toslides coated with poly L-lysine (PLL) or 3-aminopropyltriethoxysilane (APTS). Endo-genous peroxidase was inhibited with 0 5%H,02 in methanol for 10 minutes, followed bya single wash in TRIS-buffered saline (TBS).

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Antigen retrieval inimmunocywochemistry44

Five slides were placed in a plastic Goplin jar,filled with citrate buffer74 (2 1 g citric acidmonohydrate in 1 litre of distilled water, pH6-0) and covered with perforated cling film tominimise evaporation. Three Coplin jars wereplaced, evenly spaced, in a domesticmicrowave oven (Panasonic NN-6450,800W) and irradiated for five minutes atmedium power. Evaporative fluid loss wasreplaced with fresh buffer and this cycle wasrepeated up to three times. After microwaveirradiation the slides were allowed to cool toroom temperature and incubation with pri-

mary antibody was then performed at 40C for18-24 hours. In some cases a short digestionstep with trypsin (0 1% trypsin [SigmaT8128] in 0 1% CaCl2 at pH 7-8, 370C) wasincluded after microwave irradiation andbefore the application of primary antibody.Bound primary antibodies were visualisedusing a peroxidase-labelled streptavidin-biotincomplex method, with 3,3-diaminobenzidineas the chromogen.'12

All antibodies were tested with and withoutmicrowave irradiation, and the staining qual-ity was compared with that acheived using a

Figure 1 Examples ofantibodies that previouslyshowed no reactivity informalin fixed, paraffinwax embedded material,but gave good results aftermicrowave irradiation.(A) Staining quality with(right) and without (left)microwave pre-treatment:(i) MIB 1, tonsil;(ii) CD44, thyroid;(iii) CD8, tonsil.

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450~~~~~~~~~~~~~~~~~~~Cuevas,Bateman, Wilkins, Johnson, Williams, Lee, J7ones, Wright

(B) MIB 1 staining inenteropathy associated Tcell lymphoma (left),Burkitt's lymphoma(centre), and Hodgkin'sdisease (right).

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standard digestion with trypsin step (eightminutes). When lymphoid tissues were stud-ied, the proliferation marker MIB- 1 was usedas a positive control for the microwave step asthis antibody gives reliable and reproduciblestaining after microwave irradiation."Negative control sections from which primaryantibody was omitted were included in allstaining runs.

ResultsOf the 80 antibodies tested, microwave pre-treatment resulted in high quality stainingwith 20 antibodies that previously producedno staining in formalin fixed, paraffin waxembedded material (table 1) (fig 1). Anti-bodies differed in the amount of microwaveirradiation needed, but the best results wereusually obtained with two or three five minute

Table 2 Antibodies previously reactive with formalin fixed,paraffin wax embeddedtissue, but showing improvement after microwave irradiation

Antibody CD/specificity Source

Lymphocytic and myelomonocyti markersL26 CD20 DakoLNl GDw75 Biotest/GlonabBU38 CD23 Binding Site, BirmiinghamPGMI CD68 B Falini, Perugia, Italy

and DakoKi B3 B cells KielBCL-2 BCL-2 protein K Gatter, Oxford and DakoHB21 CD71 ATCCBerH2 CD30 DakoLeuMl CD15 Becton DickinsonWR14 CD43 Wessex Regional Immunology

UnitGD3 GD3 DakoPolyclonalWR18 MHC class II Wessex Regional Immnunology

UnitAdhesion moleculesBRIG 222 CD44 D Anstee, NBTS, Bristol"1HECD 1 E-cadherin S Hirohashi, Tokyo, Japan"NCC-CAD P-cadherin S Hirohashi, Tokyo, Japan"'299Hermes 1 CD44 S Jalkenen, Turku, FinlandHermes 3 CD44 S Jalkenen, Turku, FinlandMiscellaneousD07 p53 protein DakoCollagen IV Collagen type IV DakoLaminin Laminin SigmaVimnentin Viinentin Euro-PathfBio-nuclear Services

cycles. Within this group the dilutions weregenerally similar or slightly more concentratedthan those used in frozen sections.A further 21 antibodies that already stain

formalin fixed, paraffin wax embedded mater-ial showed significant improvement withmicrowave irradiation (table 2) (fig 2).Notably, the antibodies BerH2, CD3, andLeuMl produced high quality staining aftermicrowave irradiation without digestion withtrypsin that would have otherwise been neces-sary. Many of these antibodies also producedimproved staining at significantly higherworking dilutions.The remaining antibodies, of which WIRi 7,

3.9, Ki Ml., YI/82A, UGHT1i, OKT3, 4, 6,and 8, HB2., UCHL1, FIfi, TCRb1, JOVI-1and 3, PGC10, TS2177, and fibronectin arecommercially available, did not show anyimprovement after microwave treatment.With some of these we combined microwaveirradiation with a subsequent short digestionwith trypsin stage, without any improvementin staining results.

DiscussionOur study has shown that paraffin waxembedded material can be successfullyimmunostained using antibodies that werepreviously reported to stain only frozen tis-sues. Therefore., the number of antibodiesavailable for the study of archival material canbe greatly increased.

Microwave irradiation offers several advan-tages for routine immunocytochemistry.Recent studies have shown improved stainingwith several antibodies after microwave irradi-ation.61 Many of these antibodies alreadystain paraffin wax embedded tissue, and ourstudy contains fturther antibodies in this cate-gory. In some cases the staining quality ofsuch antibodies is improved; in othersmicrowave treatment may allow cost savingsto e adthroughAthe us of higherk* primaryantibody dilutions. The need for enzymatic

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Antigen retrieval in immunocytochemistry

Figure 2 Examples ofantibodies that previouslyshowed some reactivity informalin fixed, paraffinwax embedded material,and gave much improvedstaining after microwavepre-treatment. (A) Hermes3, with (right) andwithout (left) microwavepre-treatment (thyroid).(B) p53 staining inenteropathy associated Tcell lymphoma (left),Burkitt's lymphoma(centre), and Hodgkin'sdisease (right).

predigestion of sections is obviated in somecases. Many antibodies (such as CD3) alsobenefit from a reduction in background stain-ing.The adverse effects of microwave treatment

include detachment of sections from slidesduring irradiation, especially with highernumbers of cycles. This most often occurswith tissues containing prominent fibrous ele-ments (such as nodular sclerosing Hodgkin'sdisease), and under these circumstances mayoccur despite coating slides with PLL orAPTS. Background staining may also occurwith some antibodies, particularly with longerexposure times to microwave irradiation.We found that, using Coplin jars, the best

results were obtained when sections wereplaced towards one end of the slide, to ensurecontinual immersion in buffer solution duringmicrowave irradiation. With this method, amaximum of 15 slides can be irradiated dur-ing each five minute microwave cycle and thejars require repeated replenishment of evapo-rative loss. We are currently evaluating amethod for the irradiation of larger numbers

of slides in an increased volume of buffer thatmay allow antigen retrieval with a single,longer, irradiation step.The mechanism of antigen retrieval

achieved by microwave irradiation remainsobscure, but a possible explanation is thatmicrowaves disrupt the cross-linking of pro-teins in a similar way to that achieved by enzy-matic predigestion.6 At present, mostlaboratories use domestic microwave ovensfor antigen retrieval, and the characteristics ofmicrowave delivery may vary between differ-ent ovens. This could account for differencesin the reproducibility of immunostainingresults between centres.

It has been suggested that a combination ofprotease digestion and microwave treatmentmay improve the staining characteristics of asmall number of antibodies, such asimmunoglobulin light and heavy chains.19 Wecombined microwave irradiation with diges-tion with trypsin for several antibodies, butcould not improve the staining qualityachieved with microwave treatment alone.Additionally, tissue morphology was often

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Cuevas, Bateman, Wilkins, Johnson, Williams, Lee, Jones, Wright

impaired when both forms of treatment werecombined.

Based on our experience with the antibod-ies studied, antibody specificity seems to bemaintained after microwave treatment.However, the possibility of altered stainingcharacteristics should be considered wheneverantigen retrieval is used with new or pre-viously untested antibodies, and resultsshould be compared with those obtainedusing frozen section immunohistochemistry ininitial evaluation.

In conclusion, microwave irradiation repre-sents a major technical advance, enhancingtissue antigenicity while preserving morphol-ogy. It is straightforward to perform, and haswide application within research and diagnos-tic histopathology.

We thank the following for providing primary antibodies: DrD Anstee, Blood Services South West, Bristol; BectonDickinson; Dako; Dr J Gerdes, Forschungsinstitut Borstel,Borstel, Germany; Dr D Haskard, Royal PostgraduateMedical School, London; Dr S Hirohashi, Tokyo; DrJalkenen, Turku, Finland; Dr C Meijer, Amsterdam;Novocastra; Nuffield Department of Pathology, University ofOxford; Sigma; Dr J Spencer, University College, London; DrA Tutt, Tenovus, Southampton; Wessex RegionalImmunology Unit. We also thank Brian Mepham for hisadvice and technical assistance.

This work was supported in part by the Coeliac Trust.

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