Preparation of Adult Mouse Skeletal Tissue Sections for RNA In Situ Hybridization.

The RNA in situ hybridization assay is essential in many studies to evaluate gene expression in vivo. It consists of generating tissue sections and subsequently hybridizing these sections with RNA probes. Keeping RNA intact is a challenge while harvesting tissue samples, processing through embedding, sectioning them, and conditioning the sections for hybridization. These challenges are particularly strong for adult skeletal tissues due to their copious, dense, and mineralized extracellular matrices. Here, we describe a method optimized to successfully hybridize RNA species, even of low abundance, in adult mouse bone and cartilage samples. This method involves tissue fixation with paraformaldehyde, demineralization with Morse's solution and paraffin embedding, all of which can be completed in 4 days. Sections are then generated and hybridized using a 1-day standard protocol. Sections prepared using this method are compatible with immunostaining and standard staining procedures for skeletal tissues.