In situ analysis of male meiosis in C. elegans.
3D reconstruction
C. elegans
Electron microscopy
Electron tomography
Male meiosis
Microtubules
Spindles
X-chromosome
Journal
Methods in cell biology
ISSN: 0091-679X
Titre abrégé: Methods Cell Biol
Pays: United States
ID NLM: 0373334
Informations de publication
Date de publication:
2019
2019
Historique:
entrez:
22
7
2019
pubmed:
22
7
2019
medline:
30
4
2020
Statut:
ppublish
Résumé
We describe a routine method to locate cells of appropriate meiotic stages in the gonad of Caenorhabditis elegans males prior to 3D reconstruction of meiotic spindles by electron tomography. For this, serial semi-thick (300nm) sections of whole worms are pre-screened and recorded at low magnification by transmission electron microscopy. Cells of interest are identified in aligned image stacks showing the entire proximal region of male gonads at low magnification. Tilt series of selected cells are then recorded at higher magnification to reconstruct meiotic spindles of selected cells in 3D. Our approach allows a routine staging of spermatocytes without the use of anesthetics or the application of physical immobilization of worms. We also describe a modification of a previously published protocol (Muller-Reichert, Srayko, Hyman, O'Toole, & McDonald, 2007) by using polyvinylpyrrolidone (PVP) instead of bovine serum albumin (BSA) as a "filler" for specimen loading in high-pressure freezing.
Identifiants
pubmed: 31326018
pii: S0091-679X(19)30035-4
doi: 10.1016/bs.mcb.2019.03.013
pii:
doi:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
119-134Informations de copyright
© 2019 Elsevier Inc. All rights reserved.