Establishment of a standardized post-embedding method for immunoelectron microscopy by applying heat-induced antigen retrieval

We have developed a new standardized method for the post-embedding immunoelectron microscopy using the same fixation, antigen retrieval and image contrasting procedures. Tissues were fixed with 4 formaldehyde containing 2.5 mM CaCl2, 1.25 mM MgCl2 in a 0.1 M 4-(2-hydroxyethyl)-piperazineethanesulfonic acid (HEPES) buffer (pH 7.4) for 2 h and then with the same fixative composition in 0.1 M HEPES buffer (pH 8.5) overnight at room temperature. Vehicle osmolarity of fixatives was adjusted to 300-330 mOsm by adding glucose. The specimens were dehydrated with dimethylformamide on ice and embedded in LR-White resin. Ultrathin sections were heated in a 20 mM Tris-HCl buffer (pH 9.0) for 1-2 h at 95°C. After immuno-gold labeling, the sections were treated with 2 glutaraldehyde containing 0.05 tannic acid in a 0.1 M phosphate buffer (pH 5.5) for 5 min and with a 1 OsO40.1 M phosphate buffer (pH 7.4) for 5 min, and then they were double stained with uranyl acetate and lead citrate. The standardized method yielded strong and reproducible immunoreactions for soluble, membrane-bound and filamentous proteins showing an excellent image contrast without destruction of the fine structures.