A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM).
Authors
Kiseleva, ElenaAllen, Terence D
Rutherford, Sandra A
Murray, Stephen M
Morozova, Ksenia N
Gardiner, Fiona
Goldberg, Martin W
Drummond, Sheona P
Affiliation
Institute of Cytology and Genetics, Russian Academy of Science, Novosibirsk, Russia. elka@bionet.nsu.ruIssue Date
2007
Metadata
Show full item recordAbstract
This protocol details methods for the isolation of yeast nuclei from budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), immuno-gold labeling of proteins and visualization by field emission scanning electron microscopy (FESEM). This involves the removal of the yeast cell wall and isolation of the nucleus from within, followed by subsequent processing for high-resolution microscopy. The nuclear isolation step can be performed in two ways: enzymatic treatment of yeast cells to rupture the cell wall and generate spheroplasts (cells that have partially lost their cell wall and their characteristic shape), followed by isolation of the nuclei by centrifugation or homogenization; and whole cell freezing followed by manual cell rupture and centrifugation. This protocol has been optimized for the visualization of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs) and associated cyto-skeletal structures. Samples once processed for FESEM can be stored under vacuum for weeks, allowing considerable time for image acquisition.Citation
A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM). 2007, 2 (8):1943-53 Nat ProtocJournal
Nature ProtocolsDOI
10.1038/nprot.2007.251PubMed ID
17703206Type
ArticleLanguage
enISSN
1750-2799ae974a485f413a2113503eed53cd6c53
10.1038/nprot.2007.251