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Ultrastructural Localization of Antigens in the Mitotic Spindle of Budding Yeast Using High Presure Freezing and Freeze Substitution

Published online by Cambridge University Press:  02 July 2020

Thomas Müller-Reichert ,
Anthony Ashford ,
Claude Antony  and
Anthony Hyman
Thomas Müller-Reichert
Affiliation:
Max-Planck-Institut of Molecular Cell Biology and Genetics, c/o European Molecular Biology Laboratory (EMBL), Postfach 10.2209, Meyerhofstr. 1, D-69012Heidelberg, Germany Electron Microscope Laboratory, 26 Giannini Hall, University of California, Berkeley, CA94720, e-mail: tmr@uclink4.berkeley.edu
Anthony Ashford
Affiliation:
Max-Planck-Institut of Molecular Cell Biology and Genetics, c/o European Molecular Biology Laboratory (EMBL), Postfach 10.2209, Meyerhofstr. 1, D-69012Heidelberg, Germany
Claude Antony
Affiliation:
Institut Curie UMR144 CNRS, 26 rue d'Ulm, F-, 75248 Paris cedex 05, France
Anthony Hyman
Affiliation:
Max-Planck-Institut of Molecular Cell Biology and Genetics, c/o European Molecular Biology Laboratory (EMBL), Postfach 10.2209, Meyerhofstr. 1, D-69012Heidelberg, Germany
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Extract

The preservation of S. cerevisiae ultrastructure is not a trivial task, because the cell wall serves as a diffusion barrier during all steps of preparation. In addition, a high density of proteins in the cytoplasm and in the nucleus renders the visualization of cellular details difficult.

High pressure freezing in combination with freeze substitution has proven to be an advantageous technique for the preservation of the yeast mitotic spindle (1). However, the composition of the freeze substitution “cocktail” is crucial, when immunolabeling of antigens in the sample is intended. A high concentration of crosslinking agents might improve ultrastructural preservation of the sample, but will possibly destroy the antigenicity of the target proteins. In contrast, under conditions of high labeling efficiency, the ultrastructural detail may be lost. We were interested in a method, which gives a good ultrastructural preservation of the mitotic spindle and maintains the antigenicity of proteins associated with the spindle.

Type
Low Temperature Methods for Immunolabeling of Cells and Tissues
Information
Microscopy and Microanalysis , Volume 6 , Issue S2: Proceedings: Microscopy & Microanalysis 2000, Microscopy Society of America 58th Annual Meeting, Microbeam Analysis Society 34th Annual Meeting, Microscopical Society of Canada/Societe de Microscopie de Canada 27th Annual Meeting, Philadelphia, Pennsylvania August 13-17, 2000 , August 2000 , pp. 300 - 301
Copyright
Copyright © Microscopy Society of America

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References

1.Winey, M. et al., J. Cell Biol. 129 (1995) 1601CrossRefGoogle Scholar
2. This research was supported by grants to T.M.-R. from the German Science Foundation (DFG Mü 1423/1-1) and the Max-Planck Society, Germany.Google Scholar