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Immunofluorescence of Microtubule Assemblies in Amphibian Oocytes and Early Embryos

  • Thao Nguyen
  • Timothy J. Mitchison
  • Martin WührEmail author
Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 1920)

Abstract

Amphibian oocytes and embryos are classical models to study cellular and developmental processes. For these studies, it is often advantageous to visualize protein organization. However, the large size and yolk distribution make imaging of deep structures in amphibian zygotes challenging. Here we describe in detail immunofluorescence (IF) protocols for imaging microtubule assemblies in early amphibian development. We developed these protocols to elucidate how the cell division machinery adapts to drastic changes in embryonic cell sizes. We describe how to image mitotic spindles, microtubule asters, chromosomes, and nuclei in whole-mount embryos, even when they are hundreds of micrometers removed from the embryo’s surface. Though the described methods were optimized for microtubule assemblies, they have also proven useful for the visualization of other proteins.

Key words

Xenopus Amphibians Immunofluorescence Yolk clearing Development Embryos Mitotic spindle Microtubule asters Large cells 
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Notes

Acknowledgments

We thank Sean Megason, Angela DePace, Evangelos Gatzogiannis, Mike Levine, and Laurence Lemaire for usage of their microscopes. Thanks to members of the Wühr Lab for comments on the manuscript. This work was supported by grants GM39565 and 1R35GM128813 from the National Institutes of Health and Princeton University startup funding.

Supplementary material

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Supplementary Material (ZIP 25 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Thao Nguyen
    • 1
  • Timothy J. Mitchison
    • 2
  • Martin Wühr
    • 1
    Email author
  1. 1.Lewis-Sigler Institute for Integrative Genomics and Department of Molecular BiologyPrinceton UniversityPrincetonUSA
  2. 2.Department of Systems BiologyHarvard Medical SchoolBostonUSA

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