Antigen Retrieval Techniques Shi J Histochem Cytochem

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DOI: 10.1177/002215540104900801

2001 49: 931J Histochem CytochemShan-Rong Shi, Richard J. Cote and Clive R. Taylor

Antigen Retrieval Techniques : Current Perspectives

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PERSPECTIVES

Volume 49(8): 931937, 2001

The Journal of Histochemistry & Cytochemistry

http://www.jhc.org

Antigen Retrieval Techniques: Current Perspectives

Shan-Rong Shi, Richard J. Cote, and Clive R. Taylor

Department of Pathology, University of Southern California Keck School of Medicine, Los Angeles, California

SUMMARY

Development of the antigen retrieval (AR) technique, a simple method of

boiling archival paraffin-embedded tissue sections in water to enhance the signal of immu-nohistochemistry (IHC), was the fruit of pioneering efforts guided by the philosophy of

rendering IHC applicable to routine formalin-fixed, paraffin-embedded tissues for wide ap-plication of IHC in research and clinical pathology. On the basis of thousands of articles and

many reviews, a book has recently been published that summarizes basic principles for

practice and further development of the AR technique. Major topics with respect to severalcritical issues, such as the definition, application, technical principles, and further studies ofthe AR technique, are highlighted in this article. In particular, a further application of the

heat-induced retrieval approach for sufficient extraction of nucleic acids in addition to pro-teins, and standardization of routine IHC based on the AR technique in terms of a test bat-

tery approach, are also addressed. Furthermore, understanding the mechanism of the ARtechnique may shed light on facilitating the development of molecular morphology.

(J Histochem Cytochem 49:931937, 2001)

Immunohistochemistry

(IHC) has created a widefield for functional (analytical or molecular) morphol-

ogy, particularly since it has rendered immunoperoxi-dase methods applicable to routine formalin-fixed,paraffin-embedded tissues based on a series of techni-cal developments. These include increasingly sen-sitive detection systems and several pretreatmentsbefore the immunostaining procedure to recover anti-genicity masked by formalin fixation. However, thegrowing interest of pathologists who attempt furtherto expand the application of IHC staining on forma-lin-fixed, paraffin-embedded tissue sections were frus-trated by inconsistent results on fixed tissues. Morethan two decades ago, various alternative fixativeswere tried in an attempt to replace formalin in an irre-

versible chemical reaction of formalinprotein, butthey have failed, and it is likely that an ideal fixativewill never be found (Larsson 1988). On the otherhand, early IHC methods, including initial unmasking

techniques such as enzymatic digestion, failed to yield

satisfactory immunostaining for many antigens. There-

fore, the search for a simple and effective retrievalmethod has become a hot topic in IHC since the early1970s (Taylor and Cote 1994). In response to theneed for a more effective method to recover the for-malin-modified antigenicity, a high-temperature-heat-ing antigen retrieval (AR) technique, the method ofboiling paraffin tissue sections in water, was shown10 years ago to rendering IHC staining possible onarchival formalin-fixed, paraffin-embedded tissue sec-

tions (Shi et al. 1991). This AR technique was promptlyaccepted and employed by pathologists and mor-phologists worldwide, serving as a simple and effec-tive method to achieve satisfactory immunostaining

on archival tissue sections, enthusiastically describedas a revolutionary new technique and break-through in pathology (Gown et al. 1993; Boon andKok 1995).

Basic Questions: Definition, Application, andNecessary Knowledge for Beginners

In the current literature, the term antigen retrieval ispredominantly (and originally) defined as a high-tem-perature heating method to recover the antigenicity of

Correspondence to: Clive R. Taylor, MD, PhD, Dept. of Pathol-ogy, University of Southern California Keck School of Medicine,HMR 204, 2011 Zonal Avenue, Los Angeles, CA 90033. E-mail:[email protected]

Received for publication February 14, 2001; accepted May 9,2001 (1P5485).

KEY WORDS

antigen retrieval (AR)

immunohistochemistry

ISH

FISH

nucleic acid

formalin

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tissue sections that had been masked by formalin fixa-tion. Several terms were used to represent the sameAR heating method, resulting in confusion of the re-sults of literature search by Medline (Taylor and Shi2000). In contrast, the term AR was also appliedfor non-heating methods, including the enzymatic

method. Wide application of the AR technique in pa-thology and other fields of morphology has demon-strated distinct enhancement of IHC staining on archi-val formalin-fixed, paraffin-embedded tissue sectionsfor a variety of antibodies in terms of reduction of thedetection thresholds of immunostaining (increasingsensitivity) and retrieval of some antigens, such as Ki-67, MIB1, ER-1D5, androgen receptor, and many CDmarkers, which are otherwise negative in IHC. There-fore, the AR technique plays a role in amplifying thepre-detection phase, in contrast to other approachesof amplification in phases of detection (from multistepto polymeric detection systems) and post-detection

(enhanced substrate such as DAB using metal, imida-zol, CARD, anti-end product, goldsilver enhance-ment method) (Shi et al. 2000b,d).

Studies have also revealed that the AR heatingmethod could achieve satisfactory results in IHC fortissues embedded in plastic embedding media used forimmunoelectron microscopy (IEM), celloidin-embed-ded tissues, or non-embedded tissue slices, as well asin cell smear preparations fixed in non-crosslinkingfixatives (Boon et al. 2000; Evers and Uylings 2000;Shi et al. 2000c,d; Stirling 2000; Suurmeijer and Boon2000). The heat-induced retrieval approach has alsobeen applied to in situ hybridization (ISH), TUNEL

[terminal deoxynucleotidyl transferase (TdT)-medi-ated dUTP-biotin nick end-labeling], and FISH (fluo-rescence in situ hybridization)] (Bull and Harnden1999; Kitayama et al. 1999; Gu et al. 2000; Lucassenet al. 2000). For multiple IHC staining, heat was usedbetween each round of the immunostaining procedureto block crossreactivity in addition to retrieval of anti-genicity (Lan and NikolicPaterson 2000). The ARheating technique was also successfully applied forsufficient extraction of proteins from archival tissue(Ikeda et al. 1998).

Although most publications have reported satisfac-tory results of AR-IHC, several issues must be kept in

mind when this technique is used.First, not all antigen structures modified by forma-

lin can be restored using conventional AR protocols.Therefore, a test battery can be used to establish anoptimal AR protocol for certain antigens under inves-tigation (Shi et al. 2000b,d).

For a few proteins, higher temperature (boiling)may induce a negative result of IHC staining. In thiscase, a lower-temperature heating treatment or a com-bining retrieval protocol (heat and enzyme digestionor CARD) may provide better results (Cattoretti and

Fei 2000; Elias 2000; Roth et al. 2000). Recent studieshave revealed that a combination of the AR heatingmethod with a polymeric labeling two-step detectionsystem could yield a stronger signal of IHC to circum-vent the reduced sensitivity due to storage of paraffinsections (Shi et al. 1999b). Zhang et al. (2000) developed

an improved IHC method based on the AR techniqueand two-step detection reagent (EnVision; DAKO, Cam-bridge, UK) to detect enteroviral antigen in myocardiumand other tissues for laboratory diagnosis of enterovi-rus-associated diseases and for studying the mecha-nism of virus persistence in chronic myocardial disease.

Knowledge of the exact localization of a certainprotein (antigen) in tissue is critical to interpret notonly the accuracy of IHC staining results but also thereliability of AR treatment. Many excellent pioneeringstudies have given examples of approaches to scien-tific validation of the exact localization of a proteinon the basis of morphology and biochemistry or

other correlated fields (Sternberger 1978; Baskinand Stahl 1982). For this purpose, a test model sys-tem, including a fresh cell line and tissue and a for-malin-fixed, paraffin-embedded cell line and tissuethat are matched in a comparable fashion, is recom-mended to form such a basis for scientific knowl-edge of immunolocalization for certain proteins ofinterest (Hawes et al. 2000; Shi et al. 2000b). In ad-dition, control groups, including a tissue section thatis not treated by AR, are required to rule out anyfalse-positive results or altered immunostaining pat-tern (Shi et al. 2000d). Many articles recently pub-lished in this journal have described sophisticated

studies to validate the exact localization of proteinsby correlated localization of nucleic acids in cells ortissues (Cataltepe et al. 2000; Sato and Sato 2000).For example, GarciaVitoria et al. (2000) demon-strated localization of neuronal nitric oxide synthase(nNOS) in rat gastric epithelium based on compari-sons of localization of protein (IHC) and mRNA(ISH) and on polyclonal vs monoclonal antibodies,using the AR heating method for both light and elec-tron microscopy to investigate discrepancies in theliterature. Western blotting was also used to confirmnNOS protein localization in gastric epithelium.Their conclusions are convincing.

Although the AR technique is a simple method, it isnecessary to understand the factors that influence theeffectiveness of IHC staining, particularly two majorfactors, the heating conditions and the pH value of theAR solution. The heating condition represents heatingtemperature (T) and the time of heating (t), appearingto be a reverse correlation as T

t (i.e., the higher thetemperature, the shorter the heating time), to developan optimal AR protocol after a test battery approach(Chaiwun et al. 2000; Evers and Uylings 2000; Leongand Zaer 2000; Shi et al. 2000d).



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Standardization

The demand for standardization of IHC for quantita-tion is ever more pressing because of the emergence ofa new field of translational research that requiresquantitation of the differential expression of variousprognostic markers for cancer, based on retrospective

study of clinical cases with known clinical outcomes(Pertschuk and Axiotis 2000; Shi et al. 2000b). Never-theless, standardization of IHC is a huge challengethat will require great effort and more research. TheAR technique may contribute to standardization ofIHC staining for archival formalin-fixed tissue basedon the test battery approach to equalize intensity ofIHC staining under variable conditions of fixation andprocessing (Shi et al. 2000b,d). On the other hand, asatisfactory result of ARIHC must be evaluated andcompared with clinical data and other laboratorystudies, such as Western blotting and molecular bio-logical methods, to monitor the optimal AR protocol

(Pertschuk and Axiotis 2000). A model system of anantigen matrix model and the above-mentioned matchedfresh-fixed tissue model may be helpful in establish-ment of internal control and external control groupsfor standardization of IHC and to reach more accu-rate quantitative IHC (Shi et al. 2000b).

Simplifying the immunostaining procedure is one ofthe potential strategies for standardization of IHC,and optimal combination of various amplification ap-proaches to achieve a satisfactory result may be basedon the principle of simplification of all techniquesused (Kawai and Osamura 2000; Merz et al. 2000; Shiet al. 2000b; Taylor et al. 2000).

Extraction of DNA/RNA by the HeatingRetrieval Method

An interesting issue for further development of thisheat-induced retrieval approach for macromoleculesother than protein was raised by the successful appli-cation of the heat retrieval method for enhancement ofISH (Sibony et al. 1995). Oliver et al. (1997) compre-hensively compared different pretreatment regimensused to increase the sensitivity of the ISH signal in ar-chival paraffin-embedded tissue sections. The resultswere evaluated by computer-assisted image analysis,

with the conclusion that microwave retrieval regimensmay achieve the same sensitivity as that obtained onfrozen tissue sections using synthetic oligonucleotidesas probes, at the same time retaining satisfactory mor-phology on archival paraffin-embedded brain tissue.More recently, the high-temperature heating retrievalmethod has been adopted for FISH to achieve an im-proved signal, generating signal intensity comparableto that obtained by the regular enzyme digestion method.The heat-induced retrieval method is much easier tocontrol and is a simpler procedure to handle (Bull and

Harnden 1999; Kitayama et al. 1999). In our recentstudy of Her-2/neu gene amplification in urinary tractcancer, enhanced FISH signals of Her-2/neu have beenachieved using a routine heat-induced retrieval proto-col (microwave boiling of tissue sections in citratebuffer, pH 6.0, or EDTANaOH solution, pH 8.0),

with results comparable to those of enzyme digestion(Figure 1).Can the heat-induced retrieval method be used for

sufficient extraction of DNA/RNA from archivalparaffin-embedded tissue based on the achievements

Figure 1. Comparison of pretreatment protocols for fluorescence insitu hybridization (FISH) using PathVysion HER-2/CEP 17 DNA probekits (Vysis; Downers Grove, IL) on paraffin sections of a breast can-cer cell line that had been fixed in 10% neutral buffered formalinand processed routinely. Red color (rhodamine) indicates HER-2neu; green color (FITC) indicates the CEP 17. Nuclei were counter-stained with DAPI (blue color). (A) High-temperature heating wasused as the retrieval protocol (microwave heating at boiling in ci-trate buffer, pH 6.0, for 10 min). (B) Pretreatment with protease di-gestion for 15 min at 37C. A comparable signal was achieved by us-ing the simpler heating method. Original magnification 1000.



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of ISH and FISH using the heating method? Extrac-tion of DNA from archival formalin-fixed, paraffin-embedded tissue was accomplished as early as 1985without the use of the heat-induced retrieval technique(Goelz et al. 1985; Dubeau et al. 1986). After use ofPCR to amplify the signal of nucleic acids extracted

from archival tissue, heating was primarily based onthe initial heating required by the PCR technique as astep to denaturation of DNA (Shibata et al. 1988).The fact that high-temperature heating could be usedas an approach to enhance the extraction of nucleicacids or to increase the efficiency of subsequent detec-tion of a target sequence (i.e., heat-induced retrievalfor nucleic acid) was not emphasized in the literatureuntil recently. A few articles have discussed this criti-cal issue in a similar manner to the presentation of theAR technique used for IHC (Frank et al. 1996; Ma-suda et al. 1999; Coombs et al. 1999). Frank et al.(1996) compared five protocols for extracting DNA

from archival formalin-fixed, paraffin-embedded tis-sue sections and found that proteinase K digestionwith detergents followed by phenolchloroform ex-traction was not more effective than using the simpleboiling method (directly immersing the tissue sectionsin deionized, distilled, autoclaved water without a de-waxing procedure in a microcentrifuge tube and heat-ing in a boiling water bath for 8 min). Masuda et al.(1999) studied the mechanism of chemical modifica-tion of synthetic oligo-RNA by formalin to demon-strate and establish a high-temperature heating ap-proach for retrieval of RNA extracted from routinelyprocessed formalin-fixed, paraffin-embedded tissue sec-

tions. Their research is an excellent beginning in thisfield.

Studies of chemical reactions between formalde-hyde and nucleic acids also have demonstrated thatseveral basic reactions are similar to those observed informalinprotein reactions (McGhee and von Hippel1977a,b). Basic chemical reactions between nucleic ac-ids and formalin indicate that the same steps occur aswith formalinprotein reactions. The first step is anaddition reaction. All amino and imino groups ofthe nucleic acid bases can be replaced with a hy-droxymethyl (methylol) group (

CH

2

OH). As withprotein, this step is then followed by a condensation

process which is, however, a slower reaction that mayneed more than 1 week to form a stable methylenecompound. Adenine appears to be the most criticalbase in the crosslinking reaction with formalin be-cause it is rich in amino groups. In addition, the rateof the addition reaction of mono-methylol (

CH

2

OH) groups is observed to be much higher for adenine(40%) (Masuda et al. 1999). The chemical reaction ofthe condensation step is the same kind of reactionfound in the formalinprotein crosslinking process. Inany case, the methylene bridge (

CH

2

) is the major

structural element of crosslinking, and may be formedin nucleic acid, nucleic acidprotein, and proteinpro-tein interactions (Auerbach et al. 1977). Similar to theformalinprotein reaction, the overall rate of forma-lin-induced modification of DNA is dependent onboth temperature and pH values. As with protein,

most reactions of formalinDNA may be reversible,with the possible exception of the adenineformalde-hyde reaction under certain conditions.

It appears that a subtle merger of the high-tempera-ture heating AR technique for protein antigens withhigh-temperature heating retrieval of nucleic acids isfound in recent literature. Based on the similarity ofchemical reactions between proteinformalin and nu-cleic acidformalin discussed previously, it is reason-able to borrow from the heat-induced retrievaltechnique of protein to improve extraction of nucleicacids to further speed development of this new tech-nique for molecular morphology.

Mechanism of AR

A better understanding of the mechanisms of formalinfixation and antigen retrieval may be helpful in thefurther development of the retrieval technique for en-hancement of IHC and ISH, and for more efficientextraction of proteins and DNA/RNA from archivaltissue. Although the exact mechanisms of formalinfixation are still unknown, ARIHC has shed somelight on the mechanism of proteinformalin interac-tions. First of all, the success of AR has shown that themodification of protein structure by formalin is re-

versible under certain conditions, such as high-temper-ature heating or strong alkaline treatment. Several hy-potheses have been proposed to explain the possiblemechanism of ARIHC, including a recent study ofcalcium-induced modification of protein conforma-tion conducted by Morgan et al. (1994,1997). Theseauthors suggested that calcium complex formationwith proteins in formalin-fixed tissue may mask anti-gens and that the release of calcium from this cage-likecalcium complex may require a considerable amountof energy, such as high-temperature heating and cal-cium chelation by citrate. Their hypothesis was basedon a test showing that exposure of tissue to calcium

chloride (CaCl

2

) in the AR solution blocked immuno-staining of MIB1. Nevertheless, their theory is chal-lenged by subsequent observations showing that cal-cium-induced modification of protein conformationinduces loss of antigenicity for some antigens such asMIB1 and thrombospondin. This calcium-induced lossof IHC staining for certain antigens, which is consis-tent with biochemical studies in a similar fashion (Dixitet al. 1986), is independent of formalin fixation anddoes not necessarily imply a role for calcium in AR (Shiet al. 1999a).



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We have suggested a possible mechanism of the ARtechnique, i.e., the loosening or breaking of the cross-linkages induced by formalin fixation (Shi et al. 1991,1992). We believe that a critical element in the mecha-nism of ARIHC may be based on heat- or chemical-induced modification of the three-dimensional struc-

ture of formalinized protein, restoring the conditionof a formalin-modified protein structure back towardsits original structure. In other words, the mechanismof ARIHC appears to involve a re-naturation of thestructure of fixed proteins through a series of confor-mational changes, including the possible breaking (hy-drolysis) of formalin-induced crosslinkages, the entireprocess being driven by thermal energy from the heatsource (Shi et al. 1997,2000a).

The hypothesis of the heat-induced re-naturation isbased on an essential principle of immunology, i.e.,antigenantibody recognition is dependent on proteinstructure. Antibodies recognize specific epitopes local-

ized in a particular spatial configuration within theprotein molecule. This is particularly true for discon-tinuous antigenic determinants, which are composedof residues from different parts of the amino acid se-quence. A conformational change in a protein causedby formalin fixation may mask the epitope and thusaffect the antigenicity of proteins in formalin-treatedtissue. The AR method may lead to a re-naturation orat least partial restoration of the protein structure (in-duced by high-temperature heating or other non-heat-ing procedures), with re-establishment of the three-dimensional protein structure to something approachingits native condition.

The exact chemical reaction involved in this modifi-cationre-naturation mechanism is not clear. The mostlikely process that would restore part of the nativeconfiguration of a formalin-modified antigen is thehydrolysis of crosslinkages and other formalin adductsthat result from formalinprotein fixation. In supportof this hypothesis, evidence in the literature has shownthat extending the period of washing formalin-fixedtissue in water may reverse the loss of immunoreactiv-ity (Pearse 1968). Further studies concerning themechanism of ARIHC should focus on studying thealterations of protein structure that take place duringfixation and unfixation or retrieval. Recent studies

using the AR technique for IHC staining also supportthe hypothesis that hydrolysis is a critical factor in thisrestoration (re-naturation) of formalin-modified pro-tein conformation. For example, a negative immuno-staining result may be observed if archival paraffin-embedded tissue sections are heated in pure glycerinabove 100C. However, adding a 10% water solutionto this pure glycerin leads to a satisfactory positiveimmunostaining result for the same section (Beebe1999).

Biddolph and Jones (1999) demonstrated that heat-

ing is the most important factor for the AR techniqueto achieve a satisfactory result with IHC. They foundthat incubation of archival paraffin-embedded tissuesections in all AR solutions, including the citratebuffer solution of pH 6.0 at room temperature or at37C for 872 hours, failed to achieve satisfactory pos-

itive staining for a variety of antibodies tested. How-ever, this does not preclude the possibility that immer-sion of archival tissue section in water or buffersolution at room temperature for a much longer time(months or years) may achieve a positive result. Suchan outcome is consistent with our working principleregarding the overall heating conditions (heating tem-perature times time of incubation) as previously de-scribed. A much lower temperature theoretically re-quires a much longer period of incubation (Shi et al.1996,1997; Taylor et al. 1996). The fact that the pHvalue of the AR solution is another important factorinfluencing the result of ARIHC may also support

the effectiveness of hydrolysis as a basic mechanism ofAR because hydrolysis of protein is pH-dependent andformalin-induced modification of nucleic acid andprotein structures is also pH-dependent.

In conclusion, the heat-induced AR technique hasachieved wide application in pathology as well asother fields of morphology, and has provided a poten-tial approach to standardization of routine IHC forapproaching quantification of IHC, as well as more ef-fective extraction of nucleic acids from archival paraf-fin-embedded tissues. Further studies of this AR tech-nique, including the mechanism of AR, may shed newlight in molecular morphology.

Acknowledgments

Supported by the University Pathology Associates of theUniversity of Southern California and by an NIH grant 1R21 CA91249-01.

We greatly appreciate Dr Baskins invitation to write thisarticle. We wish to thank Ms Lillian Young for managementof our laboratory and Dr Yan Shi and Mr Moung Win fortechnical assistance with FISH.

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