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Titolo:
Phalloidin-eosin followed by photo-oxidation: A novel method for localizing F-actin at the light and electron microscopic levels
Autore:
Capani, F; Deerinck, TJ; Ellisman, MH; Bushong, E; Bobik, M; Martone, ME;
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
Titolo Testata:
JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY
fascicolo: 11, volume: 49, anno: 2001,
pagine: 1351 - 1361
SICI:
0022-1554(200111)49:11<1351:PFBPAN>2.0.ZU;2-X
Fonte:
ISI
Lingua:
ENG
Soggetto:
DENDRITIC SPINES; LOCALIZATION; ORGANIZATION; PLASTICITY;
Keywords:
cytoskeleton; dendritic spines; electron tomography; 3D reconstruction; Purkinje cells;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
21
Recensione:
Indirizzi per estratti:
Indirizzo: Martone, ME Univ Calif San Diego, Dept Neurosci, Natl Ctr Microscopy & Imaging Res, LaJolla, CA 92093 USA Univ Calif San Diego La Jolla CA USA 92093olla, CA 92093 USA
Citazione:
F. Capani et al., "Phalloidin-eosin followed by photo-oxidation: A novel method for localizing F-actin at the light and electron microscopic levels", J HIST CYTO, 49(11), 2001, pp. 1351-1361

Abstract

We describe a novel high-resolution method to detect F-actin at the light and electron microscopic levels through the use of the actin-binding protein phalloidin conjugated to the fluorophore eosin, followed by photo-oxidation of diaminobenzidine. This method possesses several key advantages over antibody-based labeling and structural methods. First, phalloidin binding toF-actin can tolerate relatively high concentrations of glutaraldehyde (up to 1%) in the primary fixative, resulting in good ultrastructural preservation. Second, because both eosin and phalloidin are relatively small molecules, considerable penetration of reagents into aldehyde-fixed tissue was obtained without any permeabilization steps, allowing 3D reconstructions at the electron microscopic level. By employing a secondary fixation with tannicacid combined with low pH osmication, conditions known to stabilize actin filaments during preparation for electron microscopy, we were able to visualize individual actin filaments in some structures. Finally, we show that fluorescent phalloidin can be directly injected into neurons to label actin-rich structures such as dendritic spines. These results suggest that the fluorescent phalloidin is an excellent tool for the study of actin networks at high resolution.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 12/07/20 alle ore 04:55:25