University of Southern Denmark

A single simple procedure for dewaxing, hydration and heat-induced epitope retrieval (HIER)for immunohistochemistry in formalin fixed paraffin-embedded tissue

Paulsen, I M S; Dimke, H; Frische, S

Published in:European Journal of Histochemistry

DOI:10.4081/ejh.2015.2532

Publication date:2015

Document version:Final published version

Document license:CC BY-NC

Citation for pulished version (APA):Paulsen, I. M. S., Dimke, H., & Frische, S. (2015). A single simple procedure for dewaxing, hydration and heat-induced epitope retrieval (HIER) for immunohistochemistry in formalin fixed paraffin-embedded tissue. EuropeanJournal of Histochemistry, 59(4), 303-309. [2532]. https://doi.org/10.4081/ejh.2015.2532

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A single simple procedure fordewaxing, hydration and heat-induced epitope retrieval(HIER) for immunohistochem-istry in formalin-fixed paraffin-embedded tissueI.M.S. Paulsen,1 H. Dimke,2 S. Frische11Department of Biomedicine, Universityof Aarhus2Department of Cardiovascular and RenalResearch, Institute of MolecularMedicine, University of SouthernDenmark, Odense, Denmark

Abstract

Heat-induced epitope retrieval (HIER) iswidely used for immunohistochemistry on for-malin-fixed paraffin-embedded tissue andincludes temperatures well above the meltingpoint of paraffin. We therefore tested whethertraditional xylene-based removal of paraffin isrequired on sections from paraffin-embeddedtissue, when HIER is performed by vigorousboiling in 10 mM Tris/0.5 mM EGTA-buffer(pH=9). Immunohistochemical results usingHIER with or without prior dewaxing in xylenewere evaluated using 7 primary antibodies tar-geting proteins located in the cytosol, intracel-lular vesicles and plasma membrane. No effectof omitting prior dewaxing was observed onstaining pattern. Semiquantitative analysis didnot show HIER to influence the intensity oflabelling consistently. Consequently, quantifi-cation of immune labelling intensity using flu-orescent secondary antibodies was performedat 5 dilutions of primary antibody with andwithout prior dewaxing in xylene. No effect ofomitting prior dewaxing on signal intensitywas detectable indicating similar immunore-activity in dewaxed and non-dewaxed sections.The intensity of staining the nucleus with theDNA-stain ToPro3 was similarly unaffected byomission of dewaxing in xylene. In conclusion, the HIER procedure described

and tested can be used as a single procedureenabling dewaxing, hydration and epitoperetrieval for immunohistochemistry in forma-lin-fixed paraffin-embedded tissue.

Introduction

Epitope retrieval procedures are pivotal forsuccessful application of immunohistochemi-cal techniques on formalin-fixed paraffin-

embedded tissue. Previous studies have com-prehensively shown, that the immunoreactivi-ty of formalin-fixed paraffin embedded tissuecan be restored by heating to almost boilingtemperatures. Buffers adjusted to pH 9-10appear to be the generally most effectivechoice.1 Addition of Ca2+ chelating agents fur-ther improve the retrieval of antigenic epi-topes.2 However, since no general heat inducedepitope retrieval (HIER) procedure has beenestablished, a range of buffers, exposuretimes, temperatures and pH intervals is usedin laboratories over the world.3,4 In general theHIER procedures have been incorporated inclassical staining protocols for paraffin sec-tions, which are initiated by dewaxing andrehydration of paraffin sections through theuse of xylene (or equivalent solvents) and aseries of decreasing alcohol concentrations. It has long been recognized, that the use of

HIER could have made the time-consumingdewaxing and rehydration obsolete sinceparaffin melts at 60°C and thus would be antic-ipated to disappear from the sections whenperforming HIER at 97°C.5,6 Initially thisassumption was reported to hold true in a briefletter to the editor6 and the possibility toexclude dewaxing from the protocol was laterinvestigated in an extensive study from theDako-Cytomation lab.5 This study concludedthat HIER did not remove paraffin sufficientlyand therefore a 5 min incubation in the com-mercial dewaxing product Histoclear®

(National Diagnostics, Atlanta, GA, USA) wasrecommended in the simplified immunohisto-chemistry protocol.5 The HIER procedureemployed involved immersion of slides in Dakocytomation Target Retrieval Solution pH 6.1 for30 min at 97°C followed by a continuous washin hot tap water for about 5 min. Although thiswas overall a promising simplified procedure,it was noted that an impractical gentle shakingduring the washing step was necessary toremove the paraffin, although solubilised withHistoclear®. Moreover, the initial hope thatdewaxing could be completely omitted as aseparate procedure without the loss ofimmunohistochemical quality was not fulfilled. A commercially available product

Skipdewax® (Insitus Biotechnologies,Albuquerque, NM, USA) has been reported tobe able to remove paraffin and perform HIERsimultaneously.7 In a direct comparisonbetween traditional dewaxing in xylene fol-lowed by HIER in EDTA at pH 8 and theSkipdewax one-step protocol (both treatmentsperformed for 2.5 min in a pressure-cooker),the Skipdewax protocol was found to performbetter for 3 out of the 8 tested antibodies, andequivalent for the remaining 5 antibodies.7

The exact composition of Skipdewax® is a pro-prietary of Insitus Biotechnologies. Trilogy®

(Cell Marque, Rocklin, CA, USA) is another

commercially available product with patentedcomposition claimed to combine dewaxing,rehydration, and unmasking in a single step.However, no systematic comparison betweentraditional protocols and Trilogy® with respectto immunohistochemical results can be foundin the scientific literature. In the laboratories at the former Institute of

Anatomy at Aarhus University (since 2011 partof Department of Biomedicine), we have usedtraditional xylene mediated dewaxing followedby HIER as the standard procedure for the lastapproximately 15 years. Although we havetried several HIER protocols, we have invari-ably seen the best results with boiling the sec-tions for 16 min in Tris-buffer at pH 9 supple-

European Journal of Histochemistry 2015; volume 59:2532

Correspondence: Sebastian Frische, University ofAarhus, Department of Biomedicine, Vilh. MeyersAllé 3, Universitetsparken, Bygn. 1233, 8000Århus C, Denmark. Tel. +45.87.168291 - Fax: + 45.87.167102. E-mail: sfri@biomed.au.dk

Key words: Immunolabelling; antibodies; histol-ogy; protein localization; HIER.

Contributions: IMSP, experimental design, dataacquisition (sectioning and labelling) and inter-pretation, manuscript revision and final approval;HD, experimental design, data acquisition(labelling) and interpretation, manuscript revi-sion and final approval; SF, experimental design,data acquisition (microscopy) and interpretation,manuscript drafting and final approval.

Conflict of interest: the authors declare no con-flict of interest.

Funding: the Water and Salt Research Center atthe University of Aarhus established and support-ed by the Danish National Research Foundation(Danmarks Grundforskningsfond). H. Dimke isfunded by the Novo Nordisk Foundation, theCarlsberg Foundation and the A.P. MøllerFoundation for the Advancement of MedicalScience. S. Frische is funded from theDFF:Medical Sciences and "MEMBRANES" atAarhus University.

Acknowledgments: the authors thank the histol-ogists JST, MB, JP, and HBM for inspecting thesections for semiquantitative analysis.

Received for publication: 5 May 2015.Accepted for publication: 14 October 2015.

This work is licensed under a Creative CommonsAttribution NonCommercial 3.0 License (CC BY-NC 3.0).

©Copyright I.M.S. Paulsen et al., 2015Licensee PAGEPress, ItalyEuropean Journal of Histochemistry 2015; 59:2532doi:10.4081/ejh.2015.2532

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mented with EGTA (Protocol A in this study).Results from this procedure have formed thebasis for hundreds of scientific publicationsover these years employing hundreds of differ-ent antibodies.8-11 As this procedure differs intemperature, time, heating method and pHfrom the HIER procedures investigated in pre-vious studies of simultaneous dewaxing andHIER,5-7 we decided to test if dewaxing inxylene could be excluded before HIER in ourprotocol without loss of quality of the immuno-histochemical results.

Materials and MethodsTissuesA range of archived formaldehyde-fixed

paraffin-embedded tissues were used todetermine if the omission of traditionaldewaxing and rehydration procedures beforeHIER would affect immunohistochemicalresults. The tissues originate from ourarchive of animal tissues from a number ofstudies performed in accordance with Danishlegislation on animal experiments.Accordingly, no animals were sacrificed forthis study. The tissues were all embedded in ablend of microcrystalline paraffin wax andplastic polymers with a melting point between54 and 57°C [Cellwax Plus (Polymer Added)from Cellpath Ltd., UK, local distributor:Hounisen, Aarhus, Denmark]. The formalde-hyde-fixed tissues were dehydrated throughincubation in aqueous solutions of increasingethanol concentration (2 h in 70% ethanol, 2h in 96% ethanol, 2 h in 99% ethanol) andthereafter left in xylene overnight (minimum12 h). Tissues were thereafter transferred toliquid paraffin at 60ºC and allowed to infil-trate for 2 h at this temperature. While still at60ºC, the tissues were embedded in paraffinusing a paraffin dispenser and left to cool at afreezing plate. The following tissues from rat were used: Upper and lower jaw. Fixed by perfusion

with cold 4% formaldehyde (fromparaformaldehyde powder) in 0.1 M cacody-late buffer pH 7.4. Left in fixative overnight.Decalcified in 4.13% EDTA (pH 7.4) at 4C for20 days. Archived for 5 years. Kidney. Fixed by perfusion with room tem-

perature 4% formaldehyde (fromparaformaldehyde powder) in PBS pH 7.4.Archived for 1 year. Spleen and heart. Fixed by perfusion with

cold 4% formaldehyde (from paraformalde-hyde powder) in 0.1 M cacodylate buffer pH7.2. Archived for 10 years.From the tissue-blocks, sections were cut

at a thickness of 2 µm on a Leica 2165 rotarymicrotome a few days before the immunola-

belling. The sections were collected onSuperFrost+ slides (Mentzel Gläser) andallowed to dry at room temperature for a min-imum of 2 h and subsequently heated to 60ºCfor 1 h.

Primary antibodiesTo test the hypothesis most extensively, we

chose a range of primary antibodies raised indifferent species, from different companiesand against various types of proteins (globu-lar, secretory and membrane-bound,) andknown to be located in different cellular com-partments (cytosol, intracellular vesicles andplasma membrane). We also included oneantibody recognizing a phosphorylated epi-tope. AE2a/b: a custom made rabbit polyclonal

antibody recognizing the a and b isoform ofthe anion exchanger AE2.12

Cadherin: C1821 (Sigma-Aldrich, Brøndby,Denmark), mouse monoclonal antibody rec-ognizing various cadherin isoforms.Calbindin: 10R-C106A (Fitzgerald

Industries Int., MA, USA), mouse monoclonalantibody recognizing the 28 kD isoform of thecalcium-binding protein Calbindin.CD68: MCA341R, (AbDSerotec, Kidlington,

UK), mouse monoclonal antibody recognizingthe macrophage marker CD68 from rat.13

Connexin 43: Ab11370, (Abcam,Cambridge, UK), rabbit polyclonal antibody tothe gap junction protein connexin 43. Pendrin: 2176, a custom made rabbit poly-

clonal antibody recognizing the anion-exchanger pendrin (SLC26A4).14

pNKCC2: H934, a custom made rabbit poly-clonal antibody specifically recognizing a dou-ble phosphorylated form of the furosemidesensitive sodium, potassium, 2 chloride co-transporter, NKCC2.9

Renin: ISASRREN-GF, (InnovativeResearch, Novi, MI, USA), sheep polyclonalanti-body recognizing mouse and rat renin.

Secondary antibodiesP0447: horseradish peroxidase conjugated

goat anti-mouse immunoglobulins from Dako(Glostrup, Denmark).P0448: horseradish peroxidase conjugated

goat anti-rabbit immunoglobulins from Dako. P0163: horseradish peroxidase conjugated

rabbit anti-sheep immunoglobulins fromDako.A11034: Alexa488, conjugated goat anti-

rabbit IgG from Molecular probes (cat#A11034).

Dewaxing and HIER proceduresProtocol A. Sections were dewaxed in

xylene overnight and subsequently passedthrough 3 x 10 min 99% ethanol and 2 x 10min 96% ethanol. Endogenous peroxidase

activity was then blocked with 0.35% H2O2(35% H2O2 diluted 1:99 in methanol) for 30min. Rehydration was completed by a rinse in96% ethanol, 10 min in 70% ethanol and 3rinses in ultrapure water. HIER was per-formed by placing the sections vertically inplastic slide holders (Tissue-Tek 4465,Sakura-Finetek, Værløse, Denmark) in plasticcontainers with lids (Tissue-Tek 4457,Sakura-Finetek) each containing 200 ml TEG-buffer (10 mM Tris (Sigma 7-9, cat#T1378,Sigma-Aldrich) pH 9 at RT with 0.5 mMEthylene Glycol Tetraacetic Acid (EGTA)(Titriplex 6, Merck 1.08435), maintained atroom temperature (RT). All 24 slots in eachslide holder were filled with a slide. Threeidentically filled containers were placed sym-metrically on the rotating plate in amicrowave oven (Whirlpool MD101) and heat-ed at 900 W for 6 min boiling vigorously fol-lowed by 10 min at 350W, resulting in gentlepulsatile boiling. Hereafter the sections wereleft cooling for 30 min in the TEG-buffer at RT. Protocol B. This protocol was similar to pro-

tocol A, except that dewaxing and rehydrationwas omitted. The sections were thus directlyexposed to the HIER procedure. Blockade ofendogenous peroxidase activity was per-formed for 30 min using a 8:1:1 mixture ofPBS, 35% H2O2 and methanol after HIER.Protocol C. Sections were dewaxed in

xylene overnight and subsequently passedthrough 3 x 10 min 99% ethanol and 2 x 10min 96% ethanol. Endogenous peroxidaseactivity was then blocked with 0.35% H2O2 for30 min. Rehydration was completed by a rinsein 96% ethanol, 10 min in 70% ethanol and 3rinses in ultrapure water. Hereafter, the sec-tions were placed in 10 mM TrisHCl pH 9 with0.5 mM EGTA at 60°C overnight. Although thisprocedure is less efficient with respect to tar-get retrieval than protocol A, we included it inthis test because we have found it to beinstrumental to prevent sections of bone fromdetaching the slide during HIER.Protocol D. This protocol was similar to

protocol C, except that dewaxing and rehydra-tion was omitted. The sections were thusdirectly exposed to the 60°C overnight epi-tope retrieval procedure. Blockade of endoge-nous peroxidase activity was performed as inprotocol B after the epitope retrieval proce-dure.

Immunolabelling procedure usingHRP-conjugated secondary anti-bodiesThe sections prepared through protocol A,

B, C, and D were incubated for 30 min in 50mM NH4Cl to block free aldehyde groups.Subsequently the sections were washed 3times in blocking solution (10 mM PBS con-taining 1% BSA, 0.2% gelatine, 0.05%

Technical Note

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Saponin). Antibodies were diluted in 10 mMPBS containing 0.1% BSA, 0.3% Triton X-100and the sections were incubated with the pri-mary antibodies overnight for 1 h at RT andsubsequently at 4°C overnight. The followingday, the sections were allowed to reach RTbefore they were washed 3 times in PBS con-taining 0.1% BSA, 0.2% gelatine and 0.05%Saponin followed by incubation with second-ary antibodies diluted in PBS with 0.1% BSAand 0.3% Triton X-100 for 1 h. After additional3 washes in PBS containing 0.1% BSA, 0.2%gelatine and 0.05% Saponin, peroxidase activ-ity was detected (10 min incubation) usingdiaminobenzidine (DAB) (DAB tablets pH 7,#4170, Kem-En-Tec Diagnostics, Taastrup,Denmark) in a final concentration of 1mg/mL. DAB-tablets were freshly dissolvedapproximately 20 min before use and activat-ed with 0.35% H2O2 just before use. Then sec-tions were rinsed in PBS for 3 x 10 min, twicein ultrapure water and then counterstained inMayers Hematoxylin for 2 minutes and placedin cold running tap water for 20 min.Dehydration was performed by 2x3 min incu-bation in 70%, 96% and 99% ethanol followedby 3 x 5 min in xylene before coverslips werefinally mounted using DPX (Merck,1.01979.0500).

Semiquantitative evaluation of theeffects of omitting dewaxing inxyleneTo semiquantitatively evaluate if the

immunohistochemical results of protocol Aand B (omission of dewaxing in xylene) dif-fered consistently, 4 experienced histologistsinspected 10 pairs of sections. The sections ineach pair were from the same paraffin-block(rat kidney) and processed in parallel, exceptthat one section followed protocol A and theother protocol B. Primary antibody againstpendrin and HRP-conjugated secondary anti-bodies were used. The sections wereanonymised and the pairs were made by ran-domly selecting sections prepared accordingto each protocol. Random pairs were preparedfor each histologist. The histologists wereasked if they recognized any differencebetween the immunohistochemical labellingintensity and specificity. If a difference wasrecognized, the histologist was asked toselect the section in the pair, which showedsuperior immunohistochemical labelling withrespect to intensity and specificity. If theassignment of superiority with respect tointensity and specificity is random, the num-ber of superior sections from each protocolwould follow a binomial distribution (0-hypothesis). Accordingly, we calculated theprobability of assigning superiority to thenumber of sections from each protocol by

each histologist according to the binomialdistribution. A probability <0.05 was consid-ered significant to reject the 0-hypothesis ofrandomness.

Quantification of immunolabellingusing fluorophore coupled second-ary antibodiesLabelling procedure was similar to the one

described above for HRP-conjugated secondaryantibodies, except for the following modifica-tions: i) The blockade of endogenous peroxi-dase activity was omitted. ii) ToPro3(Molecular probes, T3605) diluted 1:1000 wereadded to the solution containing secondaryantibodies for nuclear labelling. iii) Followingthe incubation with secondary antibodies only3 x wash in PBS and a final rinse in ultrapurewater were performed before coverslips weremounted using Glycergel (DAKO, C0563)

which were supplemented with 2,5%w/v 1,4-Diazabicyclo[2.2.2]octane (Merck, 803456) One section was labelled with each antibody

dilution. The labelled sections were visualizedusing a Leica Konf SL confocal microscopewith a PL APO CS 40.0x 1.25 OIL objective.Images using laser excitation at 488 and 633were obtained separately and subsequentlysuperimposed. ImageJ (http://imagej.nih.gov/ij/) was used for image analysis.Regions of interest were drawn by handaround cells which showed signal of immuno-labelling following excitation at 488 nm and inwhich the ToPro3 nuclear staining were clearlyvisible following excitation at 633 nm.Between 8 and 26 Pendrin-positive cells wereencircled in each section. Fluorescence signalsfrom Alexa488 and Topro3 were integrated ineach region of interest (ROI) and used foranalysis (see Figure 3A for illustration of theROI’s).

Technical Note

Figure 1. Comparison of immunolabelling of intracellular epitopes using protocol A(Xylene+HIER) (A,C,E,) and protocol B (HIER only) (B,D,F,). Immunolabelling forCalbindin in rat kidney (A vs B), CD68 in rat spleen (C vs D) and Renin in rat kidney(E vs F) are qualitatively similar in protocol A and B. DCT, distal convoluted tubule.Scale bars: A,B,E,F) 30 µm; C,D) 100 µm.

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ResultsImmunohistochemical results fromprotocol A and B revealed no quali-tative differencesCytoplasmic labelling was investigated by

labelling for Calbindin, which is a cytosolicprotein found (among other places) in the dis-tal nephron. The expected labelling was evi-dent in both protocol A and B (Figure 1 A,B).Further intracellular epitopes were investigat-ed by labelling for CD68 in rat spleen, whichlocalizes to the intracellular vesicular compart-ments associated with the endocytotic pathway(lysosomes) and by labelling for Renin, whichis produced and released by juxtaglomerularcells in the afferent arterioles of renalglomeruli and thus is associated with thesecretory pathway. The expected labelling forboth proteins was evident in both protocols(Figure 1 C-F). Labelling of epitopes associat-ed with the plasmamembrane was investigatedby labelling for AE2, pNKCC2 and Connexin43.AE2 is a basolateral membrane protein foundin several cell types, including osteoclasts.15

Osteoclasts in rat upper jaw labelled for AE2 inboth protocol A and B (Figure 2 A,B). An anti-body recognizing a double phosphorylated epi-tope on NKCC2 in rat kidney, previouslydescribed to be localized only on the apicalplasma membrane,9 was used to evaluate qual-itative differences in staining of apical plasmamembrane proteins and to investigate the sen-sitivity of phospho-specific epitopes to theapplied protocols. The expected labelling wasfound in both protocol A and B (Figure 2 C,D).Connexin43 is a widely distributed proteinforming gap-junctions in many tissues includ-ing ameloblasts and papillary cells in the ratenamel organ.11 The expected labelling wasseen both in protocol A and B (Figure 2 E,F).

Semiquantitative evaluation did notreveal consistent effects of HIERIn 29 of the 40 comparisons of sections

treated according to protocol A and B a differ-

ence in labelling intensity was noted (Table 1).In the sections in which a difference was rec-ognized, 2 histologists found significantly bet-ter labelling intensity in protocol A, 1 histolo-gist found significantly better labelling inten-

sity in protocol B and 1 histologist did not findany significant difference (Binomial testP<0.05). In 9 of the 40 comparisons a differ-ence in labelling specificity was noted (Table1). In the sections in which a difference was

Technical Note

Table 1. Results of the semiquantitative analysis of the results of protocol A and B. Each histologist inspected and classified 10 pairs ofsections in which one section was treated according to protocol A and one section was treated according to protocol B.

Histologist Intensity Specificity Pairs Dewaxed Non-dewaxed P Pairs Dewaxed Non-dewaxed P different superior superior Binomial different superior superior Binomial test test

1 8 7 1 0.031* 5 0 5 0.031*2 10 1 9 0.010* 0 0 0 n.a.3 8 7 1 0.031* 2 2 0 0.2504 3 0 3 0.125 2 0 2 0.250

*Statistically significant at P<0.05; n.a., not available.

Figure 2. Comparison of immunolabelling of epitopes associated with the plasma mem-brane using protocol A (Xylene+HIER) (A,C,E,) and protocol B (HIER only) (B,D,F).Immunolabelling for AE2 in osteoclasts in rat jaw (A vs B), pNKCC2 in rat kidney (C vsD) and Connexin43 in rat enamel organ (E vs F) are qualitatively similar in protocol Aand B. mTAL, medullary thick ascending limb. Scale bar: 30 µm.

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recognized, 1 histologist found significantlybetter labelling specificity in protocol B and 3histologists did not find significant differences(Binomial test P<0.05).

Quantification of immunolabellingintensity using fluorescent second-ary antibodies and nuclear stainingshowed no differences betweenprotocol A and BTo quantitatively measure the immunoreac-

tivity of sections treated according to protocolA and B, labelling of a membrane protein inindividual renal intercalated cells was quanti-fied using laser confocal microscopy. We useda rabbit polyclonal antibody directed againstPendrin, which is a membrane proteinexpressed in intercalated B cells and so-callednon-A, non-B intercalated cells.14 An exampleof the images used for quantification isshowed in Figure 3A. Quantification revealed asimilar reduction in average labelling intensityper cell following dilution of the primary anti-body while keeping the concentration of sec-ondary antibody constant using protocol A andB (Figure 3B). Similarly, quantification usinglaser confocal microscopy revealed no differ-ence between protocol A and B with respect tothe average intensity of the nuclear stainToPro3 per cell per section (Figure 3C).

Protocol C and D resulted inmarkedly weaker labelling intensitythan protocol A and B and dewax-ing was not complete in protocol DCadherin localizes to intercalated discs in

rat heart.16 As with the other epitopes investi-gated in this study, no qualitative differenceswere observed between the results obtainedusing protocol A and B (Figure 4 A,B).However, compared with protocol A and B(Figure 4 A,C), protocol C and D resulted inweaker immunolabelling (Figure 4 B,D).Similar results were obtained using other anti-bodies (data not shown). Further, dewaxingwas not complete in protocol D, resulting inheterogeneous labelling intensity (Figure 4D).

Discussion

The overall conclusion from this study isthat immunohistochemistry on paraffin-embedded tissue does not suffer significantlyfrom the omission of dewaxing in xylene,when the HIER procedure of protocol A and B(boiling in alkaline buffer supplemented withEGTA) is used. This conclusion is based onsuccessful immunodetection of epitopes locat-ed on a wide range of subcellular compart-ments in sections without dewaxing.

Technical Note

Figure 3. Quantitation of immunolabelling and nuclei staining after HIER with or with-out dewaxing in xylene. A) Rat kidney section immunolabelled for Pendrin using 1:1200dilution of the antibody + Alexa488 GAR secondary antibodies and costained for nucleiusing ToPro3; regions of interest were drawn by hand around cells, which labelled forpendrin and in which the nucleus was clearly visible (white encirclements); fluorescencesignal from Alexa488 and Topro3 were integrated in each ROI; scale bar: 30 µm. B) Theintegrated Alexa488 fluorescence signal per cell decreased as a function of dilution of theprimary antibody; no differences could be detected between the two labelling protocols.C) The average intensity per cell of ToPro3 nuclei staining did not differ between sectionsexposed to the protocol A and B.

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Semiquantitative analysis showed thatalthough 3 of 4 histologists noted significantdifferences between sections treated in proto-col A and B with respect to intensity, they didnot consistently find one protocol to producebetter results than the other. It thus seemsclear that experienced histologist are able todistinguish between sections treated accord-ing to protocol A and B, but this ability maydepend on other aspects of the labelling, e.g.,counterstain intensity or colour tone of theDAB-precipitate, which is beyond the scope ofthis study to identify. In contrast to intensity,only 1 histologist was able to distinguish sig-nificantly between the protocols with respectto labelling specificity and found protocol B toproduce the best results. Using fluorescentsecondary antibodies and quantitation by con-focal microscopy, no effect on specific signalintensity could be documented when omittingthe dewaxing and rehydration steps. This con-firms that the binding capability of the primaryantibodies does not differ between dewaxedand non-dewaxed sections. Neither did theomission of xylene mediated dewaxing affectthe staining intensity of nuclei using the DNAstain ToPro3. In some of the previous studies of the possi-

bility of omitting xylene mediated dewaxing,sections were heated for shorter time or totemperatures below the boiling point.5,7 Ourprotocol involves vigorous boiling and we thinkit is important to adhere to this procedure toobtain reproducible results. Another parame-ter, which may be of importance, is sectionthickness. Sections cut to a thickness of 2 µmwith a single tissue section on each slide wereemployed in this study, since this is our labora-tory standard. In a previous study, Yörükoglu etal.6 employed a wash in warm buffer to preventthe sections to be smeared with liquid paraffinafter combined HIER and dewaxing. In ourstudy we did not observe this phenomenon, butif thicker sections are used or several sectionsare placed on each slide we cannot excludethat the amount of paraffin to be removed mayrequire additional procedures to avoid paraffindroplets to adhere to the sections. The mostimportant parameter for paraffin removal inthis procedure seems to be the heating to tem-peratures well above the melting point ofparaffin, since in protocol D, the removal ofparaffin was incomplete. This implies, that theexact type of paraffin embedding materialcould also influence the results, since variouspolymers have different melting temperatures

and solubilities. In this study we only applied our routine

HIER solution and made no attempts to changethis in order to improve its dewaxing effects byadding detergents or less polar solvents thanwater such as diethanolamine, which is a com-ponent in the Trilogy® solution. A prior studybased on Raman-spectroscopy have showedhexane to be the most efficient dewaxingagent out of a range of solvents, including thecommercially available dewaxing, hydrationand HIER agents Trilogy® and Histoclear®.17

However, the immunoreactivity after thedewaxing protocol was not systematically test-ed, so the practical benefit of hexane asdewaxing agent remains to be shown. Thecommercially available solutions Histoclear®,SkipDewax® and Trilogy® may possess uniquebeneficial properties based on their propri-etary compositions, but this study shows thatan aqueous solution, based on standard chem-icals can remove paraffin in a HIER procedurewithout loss of immunoreactivity compared tosections dewaxed in xylene. This study thussupports a simplified procedure for immuno-histochemistry, which may help to save timeand reduce the exposure of laboratory person-nel to organic solvents.

References

1. Shi SR, Imam SA, Young L, Cote RJ, TaylorCR. Antigen retrieval immunohistochem-istry under the influence of pH using mon-oclonal antibodies. J Histochem Cytochem1995;43:193-201.

2. Shi SR, Cote RJ, Hawes D, Thu S, Shi Y,Young LL, et al. Calcium-induced modifi-cation of protein conformation demon-strated by immunohistochemistry: what isthe signal? J Histochem Cytochem 1999;47:463-70.

3. D'Amico F, Skarmoutsou E, Stivala F. Stateof the art in antigen retrieval for immuno-histochemistry. Immunol Methods 2009;341:1-18.

4. Shi SR, Shi Y, Taylor CR. Antigen retrievalimmunohistochemistry: review and futureprospects in research and diagnosis overtwo decades. J Histochem Cytochem2011;59:13-32.

5. Boenisch T. Pretreatment for immunohis-tochemical staining simplified. ApplImmunohistochem Mol Morphol 2007;15:208-12.

6. Yorukoglu K, Cingoz S. Epitope retrievaltechnique: a simple modification thatreduces staining time. AppliedImmunohistochem 1997;5:71.

7. Wong SCC, Chan JKC, Lo ESF, Chan AKC,Wong MCK, Chan CML, et al. The contribu-

Technical Note

Figure 4. Comparison of immunolabelling of cadherin in rat heart tissue using protocolA (Xylene+HIER) (A); protocol B (HIER only) (B); protocol C (Xylene+HIER at 60°C)(C); and protocol D (HIER at 60°C only) (D). No differences were detected between pro-tocol A and B. HIER at only 60°C resulted in weaker immunolabelling of Cadherin irre-spective if the sections were dewaxed in xylene (A vs C) or not (B vs D). HIER at 60°Cwithout dewaxing in xylene gave rise to heterogeneous staining intensity due to incom-plete removal of paraffin (C vs D). Asterisks indicate areas with incomplete dewaxing inD. Scale bar: 30 µm.

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[European Journal of Histochemistry 2015; 59:2532] [page 309]

tion of bifunctional SkipDewax pretreat-ment solution, rabbit monoclonal antibod-ies, and polymer detection systems inimmunohistochemistry. Arch Pathol LabMed 2007;131:1047-55.

8. de Seigneux S, Malte H, Dimke H, FrøkiaerJ, Nielsen S, Frische S. Renal compensa-tion to chronic hypoxic hypercapnia:downregulation of pendrin and adaptationof the proximal tubule. Am J Physiol RenalPhysiol 2007;292:F1256-66.

9. Dimke H, Flyvbjerg A, Bourgeois S,Thomsen K, Frøkiaer J, Houillier P, et al.Acute Growth hormone administrationinduces antidiuretic and antinatriureticeffects and increases phosphorylation ofNKCC2. Am J Physiol Renal Physiol2007;292:F723-35.

10. Praetorius J, Kim YH, Bouzinova EV,Frische S, Rojek A, Aalkjaer C, et al. NBCn1is a basolateral Na+-HCO3- cotransporter

in rat kidney inner medullary collectingducts. Am J Physiol Renal Physiol2004;286:F903-12.

11. Josephsen K, Takano Y, Frische S,Praetorius J, Nielsen S, Aoba T, et al. Iontransporters in secretory and cyclicallymodulating ameloblasts: a new hypothesisfor cellular control of preeruptive enamelmaturation. Am J Physiol Cell Physiol2010;299:C1299-307.

12. Frische S, Zolotarev AS, Kim YH,Praetorius J, Alper S, Nielsen S, et al. AE2isoforms in rat kidney: immunohisto-chemical localization and regulation inresponse to chronic NH4Cl loading. Am JPhysiol Renal Physiol 2004;286:F1163-70.

13. Dijkstra CD, Döpp EA, Joling P, Kraal G. Theheterogeneity of mononuclear phagocytesin lymphoid organs: distinct macrophagesubpopulations in the rat recognized bymonoclonal antibodies ED1, ED2 and ED3.

Immunology 1985;54:589-99.14. Kim YH, Kwon TH, Frische S, Kim J, TisherCC, Madsen KM, et al. Immunocytoche micallocalization of pendrin in intercalated cellsubtypes in rat and mouse kidney. Am JPhysiol Renal Physiol 2002;283:F744-54.

15. Josephsen K, Praetorius J, Frische S,Gawenis LR, Kwon TH, Agre P, et al.Targeted disruption of the Cl-/HCO3-exchanger Ae2 results in osteopetrosis inmice. Proc Natl Acad Sci USA 2009;106:1638-41.

16. Brüel A, Nyengaard JR. Design-basedstereological estimation of the total num-ber of cardiac myocytes in histological sec-tions. Basic Res Cardiol 2005;100:311-9.

17. Faolain EO, Hunter MB, Byrne JM,Kelehan P, Lambkin HA, et al. Raman spec-troscopic evaluation of efficacy of currentparaffin wax section dewaxing agents. JHistochem Cytochem 2005;53:121-9.

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