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Titolo:
Specific labeling of connexin43 in NRK cells using tyramide-based signal amplification and fluorescence photooxidation
Autore:
Hand, GM; Martone, ME; Stelljes, A; Ellisman, MH; Sosinsky, GE;
Indirizzi:
Univ Calif San Diego, Dept Neurosci, Natl Ctr Microscopy & Imaging Res, LaJolla, CA 92093 USA Univ Calif San Diego La Jolla CA USA 92093 ing Res, LaJolla, CA 92093 USA Univ Calif San Diego, San Diego Supercomp Ctr, La Jolla, CA 92093 USA UnivCalif San Diego La Jolla CA USA 92093 mp Ctr, La Jolla, CA 92093 USA
Titolo Testata:
MICROSCOPY RESEARCH AND TECHNIQUE
fascicolo: 3, volume: 52, anno: 2001,
pagine: 331 - 343
SICI:
1059-910X(20010201)52:3<331:SLOCIN>2.0.ZU;2-J
Fonte:
ISI
Lingua:
ENG
Soggetto:
GAP JUNCTION PROTEIN; ELECTRON-MICROSCOPY; CARDIAC MYOCYTES; PLASMA-MEMBRANE; CULTURED-CELLS; LOCALIZATION; TOMOGRAPHY; MUTATIONS; CHANNELS; IMMUNOCYTOCHEMISTRY;
Keywords:
gap junctions; membrane channels; correlative microscopy; intercellular communication;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
57
Recensione:
Indirizzi per estratti:
Indirizzo: Sosinsky, GE Univ Calif San Diego, Dept Neurosci, Natl Ctr Microscopy & Imaging Res, Room 1070,Basic Sci Bldg,Mail Code 0608,9500 Gilm, La Jolla, CA 92093 USA Univ Calif San Diego Room 1070,Basic Sci Bldg,Mail Code 0608,9500Gilm La Jolla CA USA 92093
Citazione:
G.M. Hand et al., "Specific labeling of connexin43 in NRK cells using tyramide-based signal amplification and fluorescence photooxidation", MICROSC RES, 52(3), 2001, pp. 331-343

Abstract

Imaging of gap junction proteins, the connexins, has been performed in tissue culture cells both by labeling of connexins with immunocytochemical tags and by cloning and expressing chimeras of connexins and fluorescent proteins such as Green Fluorescent Protein. These two approaches have been used to gain information about protein localization or trafficking at light microscopic resolution. Electron microscopy provides higher resolution; however, analysis of electron micrographs of unlabeled connexins has been generally limited to recognition of gap junction structures. Immunolabeling of gap junction proteins in whole cells at the electron microscopic level has beendifficult to achieve because of the fixation sensitivity of most gap junction antibodies. To obtain reasonable sensitivity, immunoperoxidase procedures are typically employed, and these suffer from relatively poor resolution. Here we describe the combination of tyramide signal amplification techniques and fluorescence photooxidation for higher resolution immunolocalization studies for correlative light and electron microscopic imaging. By using correlative microscopy, we can not only localize connexin pools or structures, but also discover what other cellular substructures interact with gap junction proteins. The use of tyramide signal amplification techniques is necessary to increase fluorescence levels that have decreased due to increased specimen fixation required to maintain cell ultrastructure. The fluorescence photooxidation technique provides a high-resolution method for stainingof proteins in cells. Unlike colloidal gold-based methods, fluorescence photooxidation allows for three-dimensional localization using high-voltage electron microscopy. Microsc. Res. Tech. 52:331-343, 2001. (C) 2001 Wiley-Liss, Inc.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 03/04/20 alle ore 11:07:32