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Electron Microscopic Immunogold Localization of Recombination-Related Proteins in Spreads of Synaptonemal Complexes From Tomato Microsporocytes

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Meiosis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 558))

Abstract

Many of the structures involved in meiotic synapsis and recombination such as synaptonemal complexes (SCs) and recombination nodules (RNs) can be resolved only by electron microscopy. Therefore, electron microscopic (EM) immunolocalization using gold-conjugated antibodies is the best way to verify whether certain proteins are components of SCs or RNs. Here, we describe (1) preparing tomato primary microsporocyte protoplasts in leptotene, zygotene, and pachytene stages; (2) hypotonically bursting the protoplasts on glow-discharged glass and plastic-coated slides to make spreads of SCs; (3) immunolabeling proteins in SCs and RNs with colloidal gold; (4) staining SC spreads for EM; and (5) transferring SC spreads on plastic films to grids for EM.

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References

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Acknowledgments

This work was supported in part by National Science Foundation grants DBI-0116076 and DBI-0421634 (S.M.S.) and MCB-0314644 (L.K.A.).

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Authors and Affiliations

  1. Department of Biology, Colorado State University, Fort Collins, CO, USA

    Stephen M. Stack & Lorinda K. Anderson

Authors
  1. Stephen M. Stack
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  2. Lorinda K. Anderson
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Editors and Affiliations

  1. Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, York Ave. 1275, New York, 10021-6007, U.S.A.

    Scott Keeney

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Stack, S.M., Anderson, L.K. (2009). Electron Microscopic Immunogold Localization of Recombination-Related Proteins in Spreads of Synaptonemal Complexes From Tomato Microsporocytes. In: Keeney, S. (eds) Meiosis. Methods in Molecular Biology, vol 558. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-103-5_10

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  • DOI: https://doi.org/10.1007/978-1-60761-103-5_10

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60761-102-8

  • Online ISBN: 978-1-60761-103-5

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