Stra te g ie s fo r Im p ro ving the lm m uno histo c he m ic a l Sta ining o f Va rio us lntra nuc le a r Pro g no stic Ma rke rs in Fo rm a lin- Pa ra ffin Se c tio ns: A nd ro g e n Re c e p to r, Estro g e n Re c e p to r, Pro g e ste ro ne Re c e p to r, ~ 53 Pro te in, Pro life ra ting C e ll Nuc le a r A ntig e n, a nd Ki- 67 A ntig e n Re ve a le d b y A ntig e n Re trie va l Te c hniq ue s zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA C LIVE R. TAYLO R, MD, PHD, SHAN-RO NG SHI, MD, BENJAPO RN C HAIWUN, MD, LILLIAN YO UNG , MT, So ASHRAF IMAM, MD, AND RICHARD J. C O TE, MD zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQ Different variations of the antigen retrieval technique using different retrieval solutions have been evaluatedfor their effectiveness in re- storingthe antigenicity of six intranuclear antigens,each of which is a potentially valuable prognosticindicator in formaliu4ixed, paraffin- embedded tissuesections.The resultsof immunohistochemical stain ing for estrogenreceptor, progesteronereceptor, androgenreceptor, ~5.9 protein, proliferating cell nuclearantigen,and Ki-67 antigen were compared following the different antigenretrievalapproaches. The strongest immunostaining signal withthe clearestbackground was ob tained by microwave heating of dewaxed parafgn sections for 10 miuutesiu 0.05 mol/L glyciueHCl (pH 3.5) or in citrate buffer so- lution (pH 6). Urea solution, distilledwater, and lead thiocyanate so- lutionyielded improvements withsome antigens, but less consistently and less impressively than glycineHCl buffer or citratebuffer. Fol- lowing antigen retrieval nuclear stabdngwas sharply defined and could be achievedconsistently in a variety of tissuesafter formalin 6xation for as long as 7 days. The durationof fixation, howwer, was an im- portantvariable;generally, the longer the fixation time the more vig- orous the retrieval procedure required. This study demonstratesthe ability to staiua variety of intrauuclear antigens, whichare not readily demonstrable otherwise, in formalin-paraffin sectionswith a high de- gree of consistency and reproducibility. The availability of methods tbat are effective in paraffin sections may facilitate studies of the possible valueof thesemarkers as prognosticindicators for predicting the response of major tumors to different forms of therapy. This study also provided insight into the basic principles of the antigen retrieval method, which may be helpful in attemptsto develop a more uuiformlystandardized techniqueapplicable to many different anti- gen systems. HUM zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA PATHOL 25~263-270. Copyright 0 1994 by W.B. Saunders Company zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA From the Department of Pathology, University of Southern Cal- ifornia School of Medicine, Los Angeles, CA. Accepted for publication September 30, 1993. Supported by University Pathology Associates of the University of Southern California. e words:immunohistochemistry, antigen retrieval, androgen re- ceptor, progesterone receptor, estrogen receptor, p53 protein, pro liferating cell nuclear antigen, Ki-67. Address correspondence and reprint requests to Clive R Taylor, MD, PhD, Department of Pathology, University of Southern California School of Medicine, HMR 204, 2011 Zonal Ave, Los Angeles, CA 90033. Copyright 0 1994 by W.B. Saunders Company 0045+3177/94/2503-0909$5.00/0 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Immunohistochemical methods are now an estab- lished, indeed essential, part of diagnostic surgical pa- thology.‘~’ The initial emphasis of immunohistochem- istry was on the identification and use of cell lineage markers, principally as a means of facilitating recogni- tion and subclassification of a wide variety of tumor types, including anaplastic tumors that proved to be dif- ficult to recognize by morphologic criteria alone. More recently the focus has changed, toward the identifica- tion of prognostic markers of potential value in pre- dicting the behavior or response to therapy of specific types of tumors. A number of the betterestablished prognostic markers are nuclear in location; these have proven par- ticularly difficult to demonstrate in routine formalin- fixed, paraffinembedded sections. For example, initial documentation of the efficacy of immunohistochemical staining for estrogen receptors (ERs) and progesterone receptors (PRs) in breast carcinoma w as Q arnered, somewhat slowly, by the use of frozen sections ,’ with all the attendant difficulties of specimen procurement and uniform rapid freezing procedures. Converting the ER and PR stains to applicability in formalin-parafhn sec- tions proved troublesome but has been achieved by a number of laboratories,‘O~ll including our own, with re- sults that compare closely with immunohistochemistry on frozen sections and with dextran-charcoal cytosol- based assays for ER and PR.” Successful application of ER and PR staining to paraffin sections requires pre- treatment of the sections by some procedure designed to “unmask” the corresponding epitope, which other- wise is rendered antigenically inert by the processes of formalin fixation and paraffin embedment. Initial stud- ies reported the use of various enzymes (eg, trypsin, DNAse) in various combinations, but such methods proved difficult to standardize even within individual laboratories and reproducibility in general practice was poor. More recently, microwave-based antigen retrieval methods have been applied with some success,” al- though, again, various modifications of the initial method have been necessary.‘3*14 263