A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM)
| Autor: | K. N. Morozova, Elena Kiseleva, Fiona Gardiner, Terence D Allen, Martin W. Goldberg, Sandra A Rutherford, Stephen M Murray, Sheona P Drummond |
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| Rok vydání: | 2007 |
| Předmět: |
Cell Nucleus
Nuclear Envelope Saccharomyces cerevisiae Cell Culture Techniques Biology Spheroplast Cell Fractionation biology.organism_classification Immunohistochemistry General Biochemistry Genetics and Molecular Biology Yeast Cell biology Cell wall Schizosaccharomyces Microscopy Schizosaccharomyces pombe Microscopy Electron Scanning Nuclear Pore Centrifugation Nuclear pore Cytoskeleton |
| Zdroj: | Nature Protocols. 2:1943-1953 |
| ISSN: | 1750-2799 1754-2189 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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