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Microtubules in Plant Cells: Strategies and Methods for Immunofluorescence, Transmission Electron Microscopy, and Live Cell Imaging

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Cytoskeleton Methods and Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1365))

Abstract

Microtubules (MTs) are required throughout plant development for a wide variety of processes, and different strategies have evolved to visualize and analyze them. This chapter provides specific methods that can be used to analyze microtubule organization and dynamic properties in plant systems and summarizes the advantages and limitations for each technique. We outline basic methods for preparing samples for immunofluorescence labeling, including an enzyme-based permeabilization method, and a freeze-shattering method, which generates microfractures in the cell wall to provide antibodies access to cells in cuticle-laden aerial organs such as leaves. We discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide chemical fixation, high-pressure freezing/freeze substitution, and post-fixation staining protocols for preserving MTs for transmission electron microscopy and tomography.

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Acknowledgements

This chapter has been supported by Austrian Science Fund (FWF) project P24242-B16 to AH and funding from the Natural Sciences and Engineering Research Council and the Canadian Institutes of Health Research to GOW. The Thieme Verlag KG is acknowledged for their kind permission to reproduce Fig. 1b, c. Figure 6a–c is reproduced with permission of the National Academy of Sciences of the United States of America, and Dr. Jennifer Lippincott-Schwartz, National Institute of Child Health and Human Development, Bethesda, MD. Elsevier Inc. for reproduction of Fig. 10.

Author information

Authors and Affiliations

  1. Department of Botany, The University of British Columbia, Vancouver, BC, Canada

    Katherine Celler, Miki Fujita & Geoffrey O. Wasteneys

  2. Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada

    Eiko Kawamura

  3. Department of Biology, University of Saskatchewan, Saskatoon, SK, Canada

    Chris Ambrose

  4. Functional Plant Biology, Institute of Botany, University of Innsbruck, Sternwartestraße 15, 6020, Innsbruck, Austria

    Klaus Herburger & Andreas Holzinger

Authors
  1. Katherine Celler
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  2. Miki Fujita
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  3. Eiko Kawamura
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  4. Chris Ambrose
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  5. Klaus Herburger
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  6. Andreas Holzinger
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  7. Geoffrey O. Wasteneys
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Corresponding author

Correspondence to Andreas Holzinger .

Editor information

Editors and Affiliations

  1. Brooklyn College, Brooklyn, New York, USA

    Ray H. Gavin

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© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Celler, K. et al. (2016). Microtubules in Plant Cells: Strategies and Methods for Immunofluorescence, Transmission Electron Microscopy, and Live Cell Imaging. In: Gavin, R. (eds) Cytoskeleton Methods and Protocols. Methods in Molecular Biology, vol 1365. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3124-8_8

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  • DOI: https://doi.org/10.1007/978-1-4939-3124-8_8

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3123-1

  • Online ISBN: 978-1-4939-3124-8

  • eBook Packages: Springer Protocols

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