Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 267-279 |
| Number of pages | 13 |
| Journal | Journal of Electron Microscopy |
| Volume | 58 |
| Issue number | 4 |
| DOIs | |
| Publication status | Published - 2009 Aug |
Fingerprint
Keywords
- Electron staining
- Heat-induced antigen retrieval
- Immunoelectron microscopy
- Post-embedding method
- Tannic acid
ASJC Scopus subject areas
- Instrumentation
Cite this
Establishment of a standardized post-embedding method for immunoelectron microscopy by applying heat-induced antigen retrieval. / Yamashita, Shuji; Katsumata, Osamu; Okada, Yasunori.
In: Journal of Electron Microscopy, Vol. 58, No. 4, 08.2009, p. 267-279.Research output: Contribution to journal › Article
}
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
Y1 - 2009/8
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
UR - http://www.scopus.com/inward/record.url?scp=68349141486&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=68349141486&partnerID=8YFLogxK
U2 - 10.1093/jmicro/dfp017
DO - 10.1093/jmicro/dfp017
M3 - Article
VL - 58
SP - 267
EP - 279
JO - Microscopy (Oxford, England)
JF - Microscopy (Oxford, England)
SN - 2050-5698
IS - 4
ER -