TY - JOUR
T1 - Establishment of a standardized post-embedding method for immunoelectron microscopy by applying heat-induced antigen retrieval
AU - Yamashita, Shuji
AU - Katsumata, Osamu
AU - Okada, Yasunori
PY - 2009/8/1
Y1 - 2009/8/1
N2 - 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.
AB - 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.
KW - Electron staining
KW - Heat-induced antigen retrieval
KW - Immunoelectron microscopy
KW - Post-embedding method
KW - Tannic acid
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U2 - 10.1093/jmicro/dfp017
DO - 10.1093/jmicro/dfp017
M3 - Article
C2 - 19332863
AN - SCOPUS:68349141486
VL - 58
SP - 267
EP - 279
JO - Microscopy (Oxford, England)
JF - Microscopy (Oxford, England)
SN - 2050-5698
IS - 4
ER -