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 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 20 mM Tris–HCl buffer (pH 9.0) for 1 h at 95�C. After immunogold labeling, the sections are treated with a mixture of tannic acid and glutaraldehyde, with OsO₄ 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.