Immunofluorescence microscopy of schizosaccharomyces pombe using chemical fixation.
Authors
Hagan, Iain MAffiliation
CRUK Cell Division Group, Cancer Research UK Manchester Institute, University of ManchesterIssue Date
2016
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Establishing the subcellular distribution of molecules of interest and the dynamics of their spatial control underpins all areas of cell and developmental biology. Although the ability to monitor the distribution of fluorescent fusion proteins has revolutionized cell and developmental biology, indirect immunofluorescence microscopy of fixed samples remains an essential complement to this approach. Immunofluorescence is often a more appropriate approach for the study of subcellular architecture. It avoids potential artifacts caused by studying fusion proteins, which might show altered function under stressful imaging conditions. Furthermore, the quantitative analysis of multiple cells in an unperturbed population by immunofluorescence invariably provides a more accurate assessment of the spatial and temporal control of a particular process than does the analysis of individual cells that is the hallmark of live-cell imaging. Parallel studies of living and fixed cells often provide complementary data sets, both of which can be considered necessary for a comprehensive understanding of molecular function. This protocol provides a method for the visualization of the Schizosaccharomyces pombe microtubule cytoskeleton by indirect immunofluorescence microscopy following chemical fixation with formaldehyde and glutaraldehyde. It includes discussion of common modifications used to monitor the distribution of other fission yeast antigens and forms a basis from which to develop protocols to localize new molecules of interest.Citation
Immunofluorescence microscopy of schizosaccharomyces pombe using chemical fixation. 2016, (7): Cold Spring Harb ProtocJournal
Cold Spring Harbor ProtocolsDOI
10.1101/pdb.prot091017PubMed ID
27371599Type
ArticleLanguage
enISSN
1559-6095ae974a485f413a2113503eed53cd6c53
10.1101/pdb.prot091017
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