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    A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM).

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    Authors
    Kiseleva, Elena
    Allen, 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.ru
    Issue Date
    2007
    
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    Abstract
    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 Protoc
    Journal
    Nature Protocols
    URI
    http://hdl.handle.net/10541/70458
    DOI
    10.1038/nprot.2007.251
    PubMed ID
    17703206
    Type
    Article
    Language
    en
    ISSN
    1750-2799
    ae974a485f413a2113503eed53cd6c53
    10.1038/nprot.2007.251
    Scopus Count
    Collections
    All Paterson Institute for Cancer Research

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