Fluorescent mRNA labeling through cytoplasmic FISH.

ABSTRACT

RNA in situ hybridization (ISH) has been widely used in cell and developmental biology research to study gene expression. Classical ISH protocols use colorimetric staining approaches, such as the assay with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP), which do not allow the implementation of multiple probe analyses and do not enable investigators to achieve cellular resolution. Here we describe a protocol to determine the presence of target cytoplasmic RNA via cytoplasmic fluorescence ISH (cFISH), an approach that renders possible the visualization of specific RNA strands from the whole tissue down to the cell. This fluorescence technique, adapted here for use in mouse embryos, enables researchers to implement multiple labeling by combining several RNA probes and/or antibodies in immuno-cFISH. Depending on the options chosen, the protocol can be completed within 2 or 3 d.