A 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 a well established technique for immunoelectron microscopy; however, many antigens cannot be detected without additional unmasking procedures. This study was undertaken to determine whether microwave oven heating, autoclaving, and pressurized boiling, which are well recognized methods of antigen retrieval for light microscopy, and simple boiling can also be used in electron microscopy. We investigated neoplastic and normal hepatocytes using a commercially available mouse monoclonal antibody against cytokeratin NO. 18 (CK 18). The tissue was fixed in paraformaldehyde/glutaraldehyde and embedded 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 before incubation with the primary antibody. The secondary antibody was gold-labeled goat anti-mouse antibody. Sections that were not heat-treated remained unlabeled, but heat-treated sections showed immunoreactivity located mainly at the cytoplasmic periphery. Some of the gold particles lay in direct or loose association with intermediate filaments, some were seen in the area of desmosomes, and some did not appear related to any structures. No difference in immunostaining was found among the four methods of heat treatment. The citrate buffer, pH 6.0, and 10 mM EDTA, pH 8.0, generated the best labeling results.
Xiao
J C
JC
Institute of Pathology, University of Tübingen, Germany.
Adam
A
A
Ruck
P
P
Kaiserling
E
E
D000941:Antigens; D006528:Carcinoma, Hepatocellular / Q000276:immunology* / Q000473:pathology; D006361:Heating; D006801:Humans; D007124:Immunoenzyme Techniques; D007633:Keratins / Q000032:analysis*; D008099:Liver / Q000166:cytology / Q000276:immunology; D008113:Liver Neoplasms / Q000276:immunology* / Q000473:pathology; D016253:Microscopy, Immunoelectron
D000941:Antigens; D007633:Keratins