Simultaneous RNA Fluorescent In Situ Hybridization and Immunofluorescent Staining of Mouse Muscle Stem Cells on Fresh Frozen Skeletal Muscle Sections

Adult muscle stem cells (MuSCs) are the key cellular source for regenerating skeletal muscle in vertebrates. MuSCs are typically identified in skeletal muscle by the expression of the paired box protein 7 (PAX7) protein. Here, we developed a combined RNA fluorescent in situ hybridization (FISH) using RNAscope technology and an immunofluorescence (IF) protocol for the simultaneous detection of <i>Pax7</i> mRNA and PAX7 protein in individual MuSCs in vivo. Interestingly, we show that while most PAX7⁺ (protein) MuSCs express <i>Pax7</i> mRNA, there is a subset of <i>Pax7</i> ⁺ (mRNA) cells that do not express PAX7 protein. Altogether, we developed a combined FISH/IF protocol that allows for the co-detection of mRNA and protein in MuSCs in vivo, a strategy that can be applied to any target gene. The functional significance of the <i>Pax7</i>-expressing subset of cells lacking PAX7 protein prior to injury remains unknown. Key features • Extensive step-by-step details for an optimized protocol combining traditional immunofluorescence with RNA fluorescent in situ hybridization (FISH) using ACDBio's RNAscope technology. • Allows for the co-detection of protein and mRNA in muscle stem cells (MuSCs) in mouse skeletal muscle tissue in vivo in ~2 days. • Validation of our protocol uncovers a subset of cells expressing <i>Pax7</i> mRNA but not PAX7 protein.