Post-embedding mammalian tissue for immunoelectron microscopy: A standardized procedure based on heat- induced antigen retrieval

Abstract

We describe a standardized method of fixation, antigen retrieval, and image contrasting for post- embedding immunoelectron microscopy. Tissues are fixed with formaldehyde solutions containing Ca2+and Mg2+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 0.5 M Tris-HCl, pH 9.0, for 1-2 h at 95°C. After immunogold labeling, the sections are treated with a mixture of tannic acid and glutaraldehyde, with OsO4solution, 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.