Nuclear Reprogramming

Boulesteix C, Beaujean N, Claire Boulesteix, Nathalie Beaujean, Boulesteix, Claire, Beaujean, Nathalie

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.