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.