The post-embedding method for immunoelectron microscopy of mammalian tissues: a standardized procedure based on heat-induced antigen retrieval.
We describe a standardized method of fixation, antigen retrieval, and image contrasting for post-embedding immunoelectron microscopy. Tissues are fixed with formaldehyde solutions containing Ca(2+) and Mg(2+) ions at pH 7.4 and then at pH 8.5. After dehydration with dimethylformamide, the specimens are embedded in LR-White resin. For antigen retrieval, ultrathin sections are heated in 20 mM Tris-HCl buffer (pH 9.0) for 1 h at 95 degrees C. After immunogold labeling, the sections are treated with a mixture of tannic acid and glutaraldehyde, with OsO(4) solution, and then double-stained with uranyl acetate and lead citrate. The standardized method yields strong and reproducible immunoreactions for many antigens showing excellent image contrast without destruction of fine structures.
Yamashita
Shuji
S
Electron Microscope Laboratory, School of Medicine, Keio University, Tokyo, Japan.
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000941:Antigens / Q000032:analysis* / Q000276:immunology; D017979:Gold Colloid / Q000737:chemistry / Q000276:immunology; D006358:Hot Temperature; D007668:Kidney / Q000166:cytology / Q000648:ultrastructure; D051379:Mice; D016253:Microscopy, Immunoelectron / Q000379:methods* / Q000592:standards; D051336:Mitochondrial Membranes / Q000276:immunology; D016610:Tissue Embedding / Q000379:methods* / Q000592:standards
D000906:Antibodies; D000941:Antigens; D017979:Gold Colloid