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% OsO4/0.1 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.