COMPARISON OF METHODS FOR HEAT-MEDIATED ANTIGEN RETRIEVAL FOR IMMUNOELECTRON MICROSCOPY - DEMONSTRATION OF CYTOKERATIN NO 18 IN NORMAL AND NEOPLASTIC HEPATOCYTES

Postembedding antigen retrieval is 21 well established technique for i mmunoelectron microscopy; however, many antigens cannot be detected wi thout additional unmasking procedures, This study was undertaken to de termine whether microwave oven heating, autoclaving, and pressurized b oiling, which are well recognized methods of antigen retrieval for Lig ht microscopy, and simple boiling can also be used in electron microsc opy, We investigated neoplastic and normal hepatocytes using a commerc ially available mouse monoclonal antibody against cytokeratin NO. 18(C K 18). The tissue was fixed in paraformaldehyde/glutaraldehyde and emb edded in Lowicryl K4M at -40 C. Ultrathin sections in various buffers were exposed to heat using one of four methods or to pronase at 37 C b efore incubation with the primary antibody, The secondary antibody was gold-labeled goat anti-mouse antibody, Sections that were not heat-tr eated remained unlabeled, but heat-treated sections showed immunoreact ivity located mainly at the cytoplasmic periphery, Some of the gold pa rticles lay in direct or loose association with intermediate filaments , some were seen in the area of desmosomes, and some did not appear re lated to any structures, No difference in immunostaining was found amo ng the four methods of heat treatment, The citrate buffer, pH 6.0, and 10 mM EDTA, pH 8.0, generated the best labeling results.