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Fluorescent Immunodetection of Epigenetic Modifications on Preimplantation Mouse Embryos

  • Claire Boulesteix
  • Nathalie BeaujeanEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1222)

Abstract

A common problem in research laboratories that study the mammalian embryo after nuclear transfer is the limited supply of material. For this reason, new methods are continually developed, and existing methods for cells in culture are adapted to suit this peculiar experimental model. Among them is the fluorescent immunodetection.

Fluorescent immuno-detection on fixed embryos is an invaluable technique to detect and locate proteins, especially nuclear ones such as modified histones, in single embryos thanks to its specificity and its sensitivity. Moreover, with specific fixation procedures that preserve the 3D shape of the embryos, immunostaining can now be performed on whole-mount embryos. Target proteins are detected by specific binding of first antibody usually nonfluorescent, and revealed with a second antibody conjugated with a fluorochrome directed specifically against the host animal in which the first antibody was produced. The result can then be observed on a microscope equipped with fluorescent detection. Here, we describe the 3D fluorescent immunodetection of epigenetic modifications in mouse embryos. This procedure can be used on nuclear transferred embryos but also on in vivo-collected, in vitro-developed and in vitro-fertilized ones.

Key words

Embryo Mouse Development Epigenetic Reprogramming Immunodetection Fluorescence Microscopy 

Notes

Acknowledgement

All the present and past members from the lab should be acknowledged for their hard work, especially Lydia Ruddick and Bénédicte Sanseau who corrected this book chapter. We are also grateful to Pierre Adenot and Renaud Fleurot for Confocal and Apotome microscopy on the MIMA2 platform (Microscopie et Imagerie des Microorganismes, Animaux et aliments). Work in the lab is supported by the REVIVE Labex.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.UMR1198 Biologie du Développement et ReproductionINRAJouy-en-Josas CedexFrance

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