PrP Immunohistochemistry: Different Protocols, Including a Procedure for Long Formalin Fixation, and a Proposed Schematic Classification for Deposits in Sporadic Creutzfeldt-Jakob Disease NICOLAS PRIVAT, 1 VE ´ RONIQUE SAZDOVITCH, 1 DANIELLE SEILHEAN, 1 JEAN-LOUIS LAPLANCHE, 2 AND JEAN-JACQUES HAUW 1 * 1 Raymond Escourolle Neuropathology Laboratory, La Salpeˆtrie`re Hospital, Pierre et Marie Curie, Paris VI University, INSERM U 360, Association Claude Bernard, 75651 Paris, France 2 Central Laboratory of Biochemistry, Lariboisie`re Hospital, Paris, France KEY WORDS diagnostic; immunohistochemistry; prion diseases; proteinase K; PrP ABSTRACT The use of immunohistochemistry on formalin-fixed and paraffin-embedded tissue has greatly improved the neuropathological diagnosis of Creutzfeldt-Jakob disease and the other subacute spongiform encephalopathies in human and animals. Two pitfalls of this technique, however, currently exist: low sensitivity after long formalin fixation and difficulties in interpreting some images.Here we review the protocols currently in use for the pretreatment of sections allowing PrP detection by immunohistochemistry. In addition, a technique useful after long for- malin fixation is reported: enzymatic digestion with proteinase K (24°C, 1/100 for 8 minutes) was employed in addition to the usual autoclaving (121°C for 10 minutes) followed by formic acid (99% for 5 minutes) and 4M guanidine thiocyanate (4°C for 2 hours). This allowed a substantial increase in the sensitivity of 3F4 immunohistochemistry on paraffin-embedded tissue, especially after prolonged formalin fixation.In addition, we suggest a simple method for classification ofPrP immunolabelling in sporadic Creutzfeldt-Jakob disease that would allow easy comparisons. Microsc. Res. Tech. 50:26 –31, 2000. © 2000 Wiley-Liss, Inc. INTRODUCTION The diagnosis of human spongiform encephalopa- thies relies on neuropathologicalexamination ofthe brain. Identification of proteinase K resistant PrP pro- tein (PrPres) in the central nervous tissue,either by Western blot or by PrP immunohistochemistry, pro- vides today the most specific diagnostic tools (Collinge and Palmer, 1996;Hauw et al., 1996;Kretzchmar et al., 1996). In contrast with Western blot, PrP immuno- histochemistry can be used on formalin-fixed and par- affin-embedded tissues. Proteinase K sensitive isoform of PrP (PrPc) seems to be a very labile protein,pre- served only after special embedding procedures (DeAr- mond et al., 1987), whereas PrPres (protease resistant isoform) withstands formalin fixation and paraffin pro- cessing. However, denaturing pretreatments are re- quired to detect PrPres by immunohistochemistry tech- niques in sections of formalin-fixed and paraffin-em- bedded tissues, and to improve the sensitivity of immunohistochemical methods. The molecular effects of these pretreatments are unknown. Formic acid (Kitamoto et al., 1987)and enzymatic digestion with proteinase K (DeArmond et al., 1987) or pepsin (Guiroy et al.,1991) were used first (Table 1). Denaturing treatments using guanidine thiocyanate (Doi-Yi et al., 1991) and heating by hydrolytic autoclav- ing (Kitamoto et al., 1992a) or microwave irradiation (Hashimoto et al., 1992) and hydrated autoclaving (Haritani et al., 1994) have also been developed. Vari- ous combinations ofthese methods were successfully tested (Hayward et al., 1994; MacDonald et al., 1996). Two multicentre prospective studies have assessed a variety of antibodies,including 3F4 (Senetekt Mary- land Heights, MO; Kascsak et al., 1987),a commer- cially available and widely used antibody, and numer- ous enhancement procedures. Whatever the antibody used, they have led to the same reliable standardized pretreatment protocol for PrPres immunohistochemis- try: hydrated autoclaving, 121°C for 10 minutes),fol- lowed by formic acid, 96% for 5 minutes, and 4M gua- nidine thiocyanate,4°C for 2 hours (Bell et al.,1997; Hegyi et al., 1997). We have developed a new method that substantially increases the sensitivity of PrP im- munohistochemistry performed on formalin-fixed par- affin embedded tissue, especially after prolonged for- malin fixation, by the addition of a supplementary step to the standardized protocol:enzymatic digestion by proteinase K (PK). MATERIALS AND METHODS Case Selection Fourteen cases were selected for this study (Table 2): 7 cases of sporadic Creutzfeldt-Jakob disease (CJD), 1 *Correspondence to: J.-J. Hauw, Laboratoire de Neuropathologie R Escourolle, GH Pitie´-Salpeˆtrie`re, 47- Bd de l’Hoˆpital, 75651, Paris Cedex 13, France. E-mail: jean-jacques.hauw@psl.ap-hop-paris.fr Received 30 October 1999; Accepted in revised form 3 January 2000 Contract grant sponsor: Programme de recherche sur les ESST et les prions, EU. MICROSCOPY RESEARCH AND TECHNIQUE 50:26–31 (2000) © 2000 WILEY-LISS, INC.