Multiplexed RNAscope and immunofluorescence on whole-mount skeletal myofibers and their associated stem cells

Skeletal muscle myofibers are large syncytial cells comprising hundreds of myonuclei, and in situ hybridization experiments have reported a range of transcript localization patterns within them. Although some transcripts are uniformly distributed throughout myofibers, proximity to specialized regions can affect the programming of myonuclei and functional compartmentalization of transcripts. Established techniques are limited by a lack of both sensitivity and spatial resolution, restricting the ability to identify different patterns of gene expression. In this study, we adapted RNAscope fluorescent in situ hybridization technology for use on whole-mount mouse primary myofibers, a preparation that isolates single myofibers with their associated muscle stem cells remaining in their niche. This method can be combined with immunofluorescence, enabling an unparalleled ability to visualize and quantify transcripts and proteins across the length and depth of skeletal myofibers and their associated stem cells. Using this approach, we demonstrate a range of potential uses, including the visualization of specialized transcriptional programming within myofibers, tracking activation-induced transcriptional changes, quantification of stem cell heterogeneity and evaluation of stem cell niche factor transcription patterns.