Multiwavelength fluorescence labeling of tissues of wholemount mouse embryos

Abstract

We present a fluorescence-based method to detect the organ and tissue sites where a specific gene is expressed in the wholemount mouse embryo. This is achieved by labeling an in situ hybridization probe which is a complementary RNA sequence to a cloned gene, and detecting the tissue-specific expression of this gene using multiple-wavelength fluorophoreconjugated antibodies directed against the label. A confocal laser scanning microscope (CLSM) is used to detect fluorescent signals of different wavelengths emanating from the same sample source. The scattering problem posed by the optically thick sample embryo is circumvented by extending the concept of space averaging and multiple staining such that by summing up the confocal images, optical noise is reduced and a higher image contrast is obtained.