Abstract
Immunolabelling electron microscopy is a challenging technique with demands for perfect ultrastructural and antigen preservation. High-pressure freezing offers an ideal way to fix cellular structure. However, its use for immunolabelling has remained limited because of the low frequency of labelling due to loss of protein antigenicity or accessibility. Here we present a protocol for immunogold labelling of the yeast Saccharomyces cerevisiae that gives specific and multiple labelling while keeping the finest structural details. We use the protocol to reveal the organisation of individual nuclear pore complex proteins and the position of transport factors in the yeast S. cerevisiae in relation to actual transport events.
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References
Tokuyasu, K. T. (1973) A technique for ultracryotomy of cell suspensions and tissues. J. Cell Biol. 8, 377–383.
van Donselaar, D., Posthuma, G., Zeuschner, D., Hmbel, B. M., and Slot, J. W. (2007) Immunogold labeling of cryosections from high-pressure frozen cells. Traffic 8, 471–485.
Mobius, W. (2009) Cryopreparation of biological specimens for immunoelectron microscopy. Ann. Anatomy, doi: 10.1016/j.aanat.2008.11.004.
Mulholland, J. and Boststein D. (2002) Immunoelectronmicroscopy of aldehyde-fixed yeast cells. In Methods in Enzymology. Guide to Yeast Genetics and Molecular and Cell Biology, Part C, Guthrie, C. and Fink, G. R., eds. Elsevier, London, vol. 351, 50–81.
McDonald, K. (2007) Cryopreparation methods for electron microscopy of selected model systems. In Methods in Cell Biology, Cellular Electron Microscopy, McIntosh, J. R. ed. vol. 79, 54–52.
McDonald, K. (1999) High pressure freezing for preservation of high resolution fine structure and antigenicity for immunolabelling. Methods Mol. Biol. 117, 77–97.
Murray, S. (2008) High pressure freezing and freeze substitution of Schizosaccharomyces pombe and Saccharomyces cerevisiae for TEM. In Methods in Cell Biology, Introduction to Electron Microscopy for Biologists, Allen, T. D. ed. vol. 88, 4–17.
Studer, D., Humbel, B. M., and Chiquet, M. (2008) Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution. Histochem. Cell Biol. 130, 877–889.
Giddings, T. H. (2003) Freeze-substitution protocols for improved visualisation of membranes in high-pressure frozen samples. J. Microsc. 212, 53–61.
Griffith, J., Mari, M., Maziere, A. D., and Reggiori, F. (2008) A cryosectioning Procedure for ultrastructural analysis and the immunogold labelling of yeast Saccharomyces Cerevisiae. Traffic 9, 1060–1072.
Terry, L. J. and Wente, S. R. (2007) Nuclear mRNA export requires specific FG nucleoporins for translocation through the nuclear pore complex. J. Cell Biol. 178, 1121–1132.
Acknowledgements
We thank to Prof. S. Wente for kindly providing the antibodies and transgenic yeast strains. This work was funded by the Biotechnology and Biological Sciences Research Council, UK grant number BB/E015735/1.
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Fiserova, J., Goldberg, M.W. (2010). Immunoelectron Microscopy of Cryofixed Freeze-Substituted Saccharomyces cerevisiae . In: Schwartzbach, S., Osafune, T. (eds) Immunoelectron Microscopy. Methods in Molecular Biology, vol 657. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-783-9_15
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DOI: https://doi.org/10.1007/978-1-60761-783-9_15
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-782-2
Online ISBN: 978-1-60761-783-9
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