Investigating the inner cell mass of the mouse blastocyst by combined immunofluorescence staining and RNA fluorescence in situ hybridization

Abstract

Immunofluorescence and RNA fluorescence in situ hybridization (FISH) methods enable the detection of, respectively, proteins and RNA molecules in single cells. Adapted to preimplantation mouse embryos, these techniques allow the investigation of transcriptional dynamics in the first embryonic and extraembryonic lineages and can circumvent the limited amount of starting material. This can as well be coupled to examination of chromatin modification, i.e., histone marks, by immunofluorescence. Here is outlined an immunofluorescence protocol combined to nascent RNA-FISH after immunosurgery of the mouse inner cell mass of the blastocyst to study early changes in transcription and/or histone marks of both primitive endoderm and epiblast cells. The method describes the different steps from coverslips and FISH probe preparation to inner cell mass isolation and immunofluorescence followed by RNA-FISH. Furthermore, this is applicable to earlier developmental stages and other mammalian species provided little technical adjustments.