title: A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM).
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.
authors: Institute of Cytology and Genetics, Russian Academy of Science, Novosibirsk, Russia. elka@bionet.nsu.ru
authors: Elena
authors: E
authors: Kiseleva
authors: Terry D
authors: TD
authors: Allen
authors: Sandra A
authors: SA
authors: Rutherford
authors: Steve
authors: S
authors: Murray
authors: Ksenia
authors: K
authors: Morozova
authors: Fiona
authors: F
authors: Gardiner
authors: Martin W
authors: MW
authors: Goldberg
authors: Sheona P
authors: SP
authors: Drummond
mesh_terms: D018929:Cell Culture Techniques; D002458:Cell Fractionation / Q000379:methods*; D002467:Cell Nucleus / Q000648:ultrastructure*; D003599:Cytoskeleton / Q000648:ultrastructure; D007150:Immunohistochemistry / Q000379:methods*; D008855:Microscopy, Electron, Scanning / Q000379:methods; D009685:Nuclear Envelope / Q000648:ultrastructure; D022022:Nuclear Pore / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000648:ultrastructure*; D012568:Schizosaccharomyces / Q000254:growth & development / Q000648:ultrastructure*