Three-Dimensional Immunofluorescence In Situ Hybridization in Preimplantation Mouse Embryos
It is now well established that three-dimensional organization of chromatin in the nucleus plays a vital role in regulating the genome. In this context, three-dimensional fluorescence in situ hybridization (3D FISH) has become a major technique used to visualize the location of DNA sequences in the nuclei and several variations of the technique have been published. In this article we describe a protocol which has been optimized for embryos to suit this peculiar experimental model without altering the 3D shape. Moreover, we will describe how to carry immuno-3D FISH to simultaneously check the localization of proteins and DNA sequences in embryos. The protocol is applicable to all preimplantation stages with several probes, thereby allowing multicolor FISH. With this method it is therefore possible to investigate nuclear localization of several genomic sequences together and apply automated imaging analyses.
Keywords3D FISH Immunostaining Development Imaging Microscopy Murine probes Mouse embryo