Journal of Immunological Methods 322 (2007) 137–142www.elsevier.com/locate/jim

Technical note

An evaluation of the immunohistochemistry benefits of boric acidantigen retrieval on rat decalcified joint tissues

Elaine Wilson a, Sonya Jackson a, Simon Cruwys b, Philip Kerry a,⁎

a Department of Pathology, AstraZeneca R&D Charnwood, Loughborough, Leicestershire, United Kingdomb Department of Discovery Bioscience, AstraZeneca R&D Charnwood, Loughborough, Leicestershire, United Kingdom

Received 24 July 2006; received in revised form 6 October 2006; accepted 24 January 2007Available online 23 February 2007

Abstract

This paper describes the evaluation and optimisation of boric acid antigen retrieval (AR) in rat joint tissue immunohistochemistry(IHC), with reference to two sample IHC targets, CD31 (PECAM-1) and Proliferating Cell Nuclear Antigen (PCNA).

Sections of buffered formalin-fixed arthritic tibial/talus joints, decalcified with EDTA, EDTA/formalin or Surgipath®Decalcifier I®, were subjected to one of a number of pre-treatments (none, 0.1% trypsin, 0.2 M acetic acid pH 7.0 or 0.2 M boricacid pH 7.0) and then immunostained for CD31 or PCNA.

Of the pre-treatment AR regimens, boric acid gave the most consistent and specific immunostaining of both antigens in jointsfrom the three different decalcification protocols. Satisfactory CD31 and PCNA staining was also achieved in EDTA decalcifiedjoints with no pre-treatment. Likewise, PCNA could be demonstrated in Surgipath® decalcified tissue without pre-treatment, albeitat slightly lower staining intensity than achieved following boric acid. The remaining decalcification/pre-treatment conditions wereunsatisfactory for IHC of the two antigens investigated because of lack of staining, non-specific staining or consistent loss ofsections from the slides.

Boric acid pre-treatment provides a valuable alternative low temperature AR method where conventional heat-mediated ARmethods are normally required but cannot be used due to tissue type.© 2007 Elsevier B.V. All rights reserved.

Keywords: Boric acid; Low temperature antigen retrieval; Immunohistochemistry; CD31 (PCAM-1); Proliferating Cell Nuclear Antigen (PCNA);Joint tissues

1. Introduction

The benefits of boric acid antigen retrieval (AR) insoft tissues have been known for some years, with theearliest located reference reporting impressive results foroestrogen and progesterone receptor immunohistochem-

⁎ Corresponding author. AstraZeneca R&D Charnwood, BakewellRoad, Loughborough, Leicestershire LE11 5RH, United Kingdom.Tel.: +44 1509 64 4188.

E-mail address: philip.kerry@astrazeneca.com (P.J. Kerry).

0022-1759/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.jim.2007.01.020

istry (IHC) in human breast tissue (Peston and Shousha,1998). These authors identified certain limitations,including the ability of EDTA to diminish immunostain-ing intensity when added to antigen retrieval solutions.This caveat may help explain the absence of informationin the subsequent literature relating to boric acid AR anddecalcified joint tissue IHC in human or other species.

In our laboratory, difficulties have been experiencedin staining for a number of antigens, including CD31(PECAM-1, a specific marker of endothelial cells;Middleton et al., 2005) and Proliferating Cell Nuclear

138 E. Wilson et al. / Journal of Immunological Methods 322 (2007) 137–142

Antigen (PCNA), in arthritic rat joints routinely fixed inneutral buffered formalin and decalcified in EDTA/formalin. In particular, high temperature AR (i.e. pres-sure cooking or microwave antigen retrieval) has beenproblematical because of related lifting or loss of hardtissue sections from charged (SuperFrost® Plus) orsilane-treated slides. Other laboratories have reportedsimilar problems of this nature (see Shi et al., 1991). Theuse of other decalcification reagents for specific needs(e.g. speed of study turn around) has complicated ourIHC procedures still further.

In an attempt to find a viable low temperature ARalternative for use on decalcified joints, an evaluationexercise has been undertaken in which boric acid wascompared with some of our existing battery of ARmethods in joint tissues decalcified by one of threedifferent regimens. CD31 and PCNAwere the two IHCtarget examples chosen to illustrate the successfulapplication of boric acid AR in joint tissue and are ofparticular interest to this laboratory as markers ofangiogenesis (CD31) and synovitis/synovial hyperpla-sia (PCNA) in models of rodent joint inflammation.

2. Experimental

2.1. Animal tissues

The tissues for examination were taken from sevenstudies all involving the streptococcal cell wall-inducedarthritis model in the rat. Briefly, female Lewis rats weresensitised by intra-articular injection of 5 μg streptococ-cal cell wall (SCW) into the tibial/talus joint and 21 dayslater challenged with 100 μg SCW i.v. Experiments wereterminated on day 5 or 6 after challenge. All experimen-tal procedures were conducted in accordance with therequirements of the Animals (Scientific Procedures) Act1986 of the United Kingdom. The joints were fixed in10% neutral buffered formalin (NBF; Pioneer ResearchChemicals, Code No. PRC/R/4) for a minimum of10 days, a time determined empirically to give optimalpreservation of all tissue components of the jointincluding the bone marrow compartment.

2.2. Decalcification procedures

Following fixation, the joints were decalcified by oneof the following three methods. The conditions for thethree regimens have been derived empirically. Duringthe decalcification process, the tissue was frequentlytested with a sharp implement to determine the earliestpoint at which they could be satisfactorily processed tosection. It was important to minimise the time the joints

spent in the relatively harsh decalcification solutions inthe interests of optimal tissue and antigen preservation.

2.2.1. EDTA (5.17% w/v; disodium salt)/formaldehyde(3.8% w/v) in deionised water, pH 6.8–7.2 (PioneerResearch Chemicals)

The joints were decalcified at 50 °C for 39 days with5 weekly changes of the decalcification solution. Whendecalcification was complete, the tissues were washed inwater for 6 h before processing.

2.2.2. EDTA (12% w/v; disodium salt) in deionisedwater, pH 6.8–7.2 (Pioneer Research Chemicals)

The joints were decalcified for 15 days with onechange of solution after 7 days. When decalcificationwas complete, the tissues were washed in water for 6 hbefore processing.

2.2.3. Decalcifier I® (Surgipath® Europe Ltd.); con-taining formaldehyde (10% w/v), formic acid (8% w/v)and methanol (1% w/v)

The joints were decalcified for 10 days at roomtemperature with no changes of solution. Tissues werenot washed before processing.

2.3. Immunohistochemistry

After decalcification, all tissues were processedovernight and embedded into paraffin wax. Sections,4 μm, were floated on to charged glass slides (Super-Frost® Plus, VWR International), dried for 48 h at37 °C, dewaxed and rehydrated to deionised water.

2.3.1. Pre-treatment/ARTissue sections were subjected to one of the

following pre-treatment steps: no pre-treatment (NPT),incubation with 0.1% w/v trypsin (Trypsin 250, DIFCOBecton Dickinson) in 0.1% w/v CaCl2·2H2O, pH 7.8 at37 °C for 30 min, incubation with 0.2 M acetic acid, pH7.0 at 60 °C for 17 h, or incubation with 0.2 M boricacid, pH 7.0 at 60 °C for 17 h. Following AR all sectionswere washed in deionised water. Acetic acid and boricacid were selected for inclusion in this study based onthe positive data reported by Peston and Shousha(1998). For comparison trypsin digestion was includedas a low temperature AR method (see Cattoretti et al.,1993), as routinely used in our laboratory.

2.3.2. Immunostaining for CD31 and PCNAEndogenous peroxidase was blocked with 3% v/v

hydrogen peroxide in methanol for 10 min and sectionswashed firstly in deionised water and then in PBS (Sigma)

Table 1Summary of IHC results comparing three different decalcificationmethods and five different pre-treatment regimens

Antigen retrieval method Decalcification method

EDTA DecalcifierI®

EDTA/formalin

(a) CD31 (PECAM-1)None +++ − −None+overnight incubation with

primary antibody (4 °C)+++ NSS −

0.1% w/v Trypsin pH 7.8 − − −0.2 M Acetic acid pH 7.0 X X X0.2 M Boric acid pH 7.0 +++ ++ ++

(b) Proliferating Cell Nuclear Antigen (PCNA)None +++ ++ NSSNone + overnight incubation with

primary antibody (4 °C)++ NSS NSS

0.1% w/v Trypsin pH 7.8 − − −0.2 M Acetic acid pH 7.0 X X X0.2 M Boric acid pH 7.0 +++ +++ +++

Key: (−)=No staining; (+), (++), (+++)=subjective scores indicativeof degree of specific immunostaining intensity/target antigen localisa-tion; (NSS)=non-specific staining, a measure of ectopic expressionand diffuse localisation of signal; (X)=sections not present forevaluation (i.e. detached from slides during pre-treatment).

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with 0.05% v/v Tween 20, pH 7.4 (PBS/Tween). Non-specific Ig-binding sites were blocked with either 20% v/vnormal horse serum (Dako) for CD31 and 20%v/v normalrabbit serum (Dako) for PCNA in PBSwith 1%w/v BSA,pH 7.4 (Sigma) for 20 min at room temperature.

Sections were incubated with either mouse anti-ratCD31 (10 μg/ml; Serotec, Clone TLD-3A12, Code No.MCA1334G) for 15 min (negative control=concentra-tion-matched Ig of the same isotype; mouse IgG1,10 μg/ml; Dako, Clone DAK-G01, Code No. X0931) ormouse anti-human PCNA (0.45 μg/ml; Vector, ClonePC10, Code No. VP-P980) for 60 min (negative con-trol=concentration-matched mouse IgG2a, 0.45 μg/ml;Dako, Code No. X0943). All antibodies were diluted inPBS with 1% w/v BSA, pH 7.4 (Sigma).

Following a PBS/Tween wash, CD31 sections werelabelled with biotinylated horse anti-mouse (1.7 μg;Vector, Code No. BA-2001) for 15 min at roomtemperature and PCNA sections labelled with biotiny-lated rabbit anti-mouse (2.9 μg; Dako, Code No. E0465)for 20 min at room temperature.

The IHC signal was visualised with the TyramideSignal Amplification (TSA) kit (Perkin-Elmer), and inthe case of CD31 also with the Catalysed SignalAmplification (CSA) kit (Dako, K1500) for comparisonpurposes.

A Lab Vision Autostainer 480 was used to stan-dardise the staining protocol for both CD31 and PCNA.Following immunostaining all sections were counter-stained with Gills Haematoxylin (Pioneer ResearchChemicals Ltd.), dehydrated, cleared and mounted innon-aqueous medium (CV; Leica).

3. Results

The CD31 and PCNA immunostaining results aresummarised in Table 1(a) and (b). Pre-treatment withtrypsin failed to elicit an IHC signal for either CD31 orPCNA in tissue decalcified in EDTA, EDTA/formalin orSurgipath® Decalcifier I®. All sections pre-treated with0.2 M acetic acid pH 7.0 at 60 °C for 17 h becamedetached from the slides and thus were not availablefor evaluation. The negative isotype controls gave noimmunoreactivity for either antibody.

Pre-treatment with 0.2 M boric acid gave the mostconsistent CD31 staining with both EDTA/formalin andDecalcifier I® decalcification protocols. Fig. 1 comparesthe CD31 signal obtained with boric acid pre-treatmentagainst no pre-treatment. Fig. 1(A)–(D) are micrographsof representative serial sections of an inflamed/hyper-plastic synovium from a buffered formalin-fixedarthritic rat joint decalcified in EDTA/formalin and

clearly demonstrate the advantages of boric acid pre-treatment (A) and (C) versus NPT (B) and (D) indelineating vascular endothelial cells, the target forCD31 expression. The CSA kit used to visualise CD31immunoreactivity in (A) and (B) gave a cleaner butslightly less pronounced signal than that obtained withthe TSA detection system ((C) and (D)). The TSA kitgenerally produces more of a background wash than theCSA system in our laboratory but, in the case of CD31,this was insufficient to significantly interfere with theinterpretation of endothelial staining.

Boric acid pre-treatment was also successful as aCD31 AR method on sections from buffered formalin-fixed arthritic joints decalcified with Surgipath®Decalcifer I®. This pre-treatment gave intense specificCD31 staining of endothelium in vessels of hyperplasticsynovium (Fig. 1(E)), which was not the case in theabsence of pre-treatment (NPT; Fig. 1(F)). The CSA kitwas used to visualise the IHC signal as demonstrated inthese representative micrographs.

The results demonstrated above essentially apply tothe articular surfaces, synovium and surrounding softtissues, our principal areas of research interest. A lessintense specific CD31 staining of vascular endotheliumwithin the bone marrow compartment was obtained withboth EDTA/formalin and Decalcifier I® after boric acidpre-treatment.

Fig. 1. CD31 IHC comparison of tissue sections which were pre-treated with boric acid (A), (C) and (E) or which received no pre-treatment (B),(D) and (F). Serial sections of inflamed synovium (A)–(D) showing evidence of angiogenesis from a buffered formalin-fixed arthritic rat jointdecalcified in EDTA/formalin; the CSA kit was used to visualise the CD31 signal in (A) and (B), the TSA detection system for (C) and (D). Adjacentserial sections (E) and (F) of hyperplastic synovium from a buffered formalin-fixed arthritic joint decalcified with Surgipath® Decalcifer I®; the CSAkit was used to visualise the CD31 signal. Magnification bar=100 μm.

140 E. Wilson et al. / Journal of Immunological Methods 322 (2007) 137–142

Sections of joint decalcified in EDTA alone gavepositive CD31 staining of synovial and surrounding softtissue vascular endothelium in the absence of any pre-treatment (not illustrated). However, the bone marrowcompartment did not show consistent endotheliumstaining with boric acid or NPT.

NPT/overnight incubation with the CD31 antibodygave non-specific staining with Surgipath® Decalcifier

I® and no staining following EDTA/formalin decalcifi-cation (not illustrated).

In the absence of any pre-treatment, PCNA immunos-taining of sections from EDTA/formalin or from Surgi-path® Decalcifier I® decalcified joints gave diffuse andpatchy non-specific cytoplasmic and interstitial staining(Fig. 2(B), (D) and (F). Boric acid pre-treatment produceda marked improvement in the quality of PCNA staining,

Fig. 2. PCNA IHC comparison of tissue sections which were pre-treated with boric acid (A), (C) and (E) or which received no pre-treatment (B),(D) and (F). Adjacent serial sections of the talus bone (A) and (B) and of the synovium (C) and (D) from the same buffered formalin-fixed arthritic ratjoint decalcified in EDTA/formalin. (A) and (B) show a granulation reaction involving the superficial cartilage and the adjacent trabecular space;(C) and (D) show a heavily inflamed and hyperplastic synovium. Adjacent serial sections (E) and (F) of hyperplastic synovium from a bufferedformalin-fixed arthritic joint decalcified with Surgipath® Decalcifer I®. The TSA detection system was used to visualise the IHC signal in all theimages shown. Magnification bar=100 μm.

141E. Wilson et al. / Journal of Immunological Methods 322 (2007) 137–142

with the immunoreactivity clearly confined to the nuclei ofthose cells undergoing cell division, more specifically,those cells in S-phase and, to a lesser extent, those in M-phase of the mitotic cycle (Fig. 2(A), (C) and (E)).

NPT with overnight incubation with the PCNAantibody gave diffuse non-specific staining with both

EDTA/formalin and Surgipath® Decalcifier I® decalci-fication (not illustrated).

Sections of joints decalcified in EDTA alone gavepositive PCNA staining in the articular joint, synoviumand surrounding soft tissues in the absence of any pre-treatment (not illustrated). Only inconsistent and weak

142 E. Wilson et al. / Journal of Immunological Methods 322 (2007) 137–142

PCNA staining was seen in bone marrow with both NPTand boric acid.

4. Discussion

The investigation by Peston and Shousha on the ARproperties of five acids (acetic, oxalic, boric, formic andcitric acids) demonstrated that low temperature AR withacetic or boric acid (both used as 0.2M solutions adjustedto pH 7.0 with 1 M NaOH) was of benefit in the IHClocalisation of oestrogen and progesterone receptors informalin-fixed paraffin sections of human breast tissue.Acetic acid gave the greatest intensity of immunostaining,but with some loss of tissue preservation, whereas boricacid gave good immunoreactivity whilst preserving tissuemorphology (Peston and Shousha, 1998).

These data suggested that low temperature boric acidAR might offer wider benefits for IHC in other species(e.g. rat), other tissues, including those requiringdecalcification, and for antigen targets other than steroidreceptors. Subsequent investigations in our laboratoryhave now confirmed the benefits of boric acid pre-treatment when applied to a small number of IHC targetsin sections of buffered formalin-fixed rat joints subjectedto three different decalcification regimens (EDTA,EDTA/formalin and Surgipath® Decalcifier I®).

Boric acid AR in combination with EDTA/formalindecalcification has proved particularly useful in studyingmarkers of inflammatory joint disease. This decalcifica-tion protocol applied to buffered formalin-fixed jointtissues has been in use as the method of choice in ourlaboratory for some years. It provides better tissuepreservation than with EDTA alone and bone marrowmorphology in particular is greatly improved.With EDTAalone the immunostaining for both targets in the bonemarrow is much weaker and patchy, suggesting thatformaldehyde could be protecting the antigen sites fromthe damaging effects of the decalcification reagent(s).However, a significant downside to adding formalin to thedecalcification solution has been increased difficulties inimmunostaining for a number of antigens.

Our battery of AR methods routinely employed in anattempt to overcome these difficulties has given mixedresults. High temperature AR by pressure cooker ormicrowaves (Shi et al., 1997), frequently of value withsoft tissue IHC, has proved problematical when dealingwith hard tissues, the extreme conditions tending to causelifting or complete loss of sections from microscopeslides. This was similar to the problem encountered withacetic acid pre-treatment presented above. Trypsin pre-treatment, again as indicated above, has not provided asolution to problems of AR in joint tissue.

Themechanismbywhich boric acid acts as anARagentis not known but there has been speculation that it worksthrough acid hydrolysis, even with the reagent adjusted topH 7.0, and that its activity may also be related to itsproperties as a Lewis acid (Peston and Shousha, 1998).

Boric acid AR has been used in this laboratory withsuccess with other antigens in human articular cartilage;these antigens include Growth/Differentiation Factor-5(GDF-5) and Transforming Growth Factor βII(TGFBRII). Boric acid has also proved of value in thevalidation of several other pharmacological receptors aspotential disease targets in human and rodent tissues butunfortunately it has not been possible to include thesedata for reasons of commercial sensitivity.

In summary, this communication describes a formalcomparison of three selected AR procedures on jointtissue decalcified by three different protocols, with twoIHC markers of relevance to the study of inflammatoryjoint disease. The study was not intended to be anexhaustive comparison. The results of this work indicatethat boric acid pre-treatment can provide a valuablealternative low temperature AR method in situationswhere conventional heat mediated AR methods are nor-mally required but cannot be used due to tissue type.

Acknowledgements

We are grateful to Dr. Martyn Foster and Mrs. IsabelClamp for helpful discussions during the course of thiswork.

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