Methods in laboratory investigation. Immunoelectron microscopic methods for demonstration of antigens on normal human melanocytes and other epidermal cells.
Different procedures for fixation and processing were evaluated in order to examine the antigenic profile of melanocytes and other epidermal cells for immunoelectron microscopy. For this purpose the monoclonal antibodies anti-HLA-A, B, C, anti-HLA-DR, anti-T6, and the melanoma-associated monoclonal antibody NKI /C-3 were used as markers. Fixation with periodate-lysine-paraformaldehyde yielded better antigenic and ultrastructural preservation than 3% paraformaldehyde or picric acid-paraformaldehyde did. Skin was further processed by five different methods: (a) 15-micron frozen sections; (b) 75-micron agar-embedded, tissue chopper sections; (c) 15-micron polyethylene glycol-embedded sections; (d) epidermal cells in suspension; and (e) epoxy sections (postembedding staining) were prepared for the immunoperoxidase procedure. Antigenicity was best preserved in the cell suspension method and somewhat less, but with a similar staining distribution, with the first three methods. Staining with the polyethylene glycol-embedded sections was only achieved if they were left free-floating in buffer; no staining was observed when the sections were mounted on glass slides and left to dry overnight at 37 degrees C. Epidermal cells remained unreactive in the postembedding method, even after etching. Ultrastructural preservation of the agar-embedded sections and the cells in suspension was superior to the other preembedding methods. Melanocytes mostly showed moderate staining for HLA-A, B, C and slight staining for the antigen that is recognized by NKI /C-3. The latter was further demonstrated on Langerhans cells and indeterminate cells which also expressed HLA-A, B, C, T6, and HLA-DR antigens.
D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000032:analysis*; D000945:Antigens, Differentiation, T-Lymphocyte; D000954:Antigens, Surface / Q000032:analysis; D000078404:Epidermal Cells; D006680:HLA Antigens / Q000032:analysis; D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II / Q000032:analysis; D006801:Humans; D066298:In Vitro Techniques; D008544:Melanocytes / Q000276:immunology*; D008854:Microscopy, Electron / Q000379:methods
D000911:Antibodies, Monoclonal; D000941:Antigens; D000945:Antigens, Differentiation, T-Lymphocyte; D000954:Antigens, Surface; D006680:HLA Antigens; D006684:HLA-DR Antigens; D000949:Histocompatibility Antigens Class II