Methods for In situ Quantification of Mitochondrial Morphology in Muscle and Terminal Schwann Cells of Mice

Mitochondrial network morphology is widely used as an indicator of cellular health; however, quantifying mitochondrial architecture within intact tissues remains technically challenging. Terminal Schwann cells (tSCs), which are essential for the maintenance and regeneration of neuromuscular junctions, are particularly difficult to analyze in situ due to their anatomical location and sensitivity to tissue disruption. This protocol describes a reproducible approach for labeling and quantifying three-dimensional mitochondrial network morphology in whole-mount skeletal muscle and in tSCs in mice using standard confocal microscopy. The method employs in vivo delivery of a membrane potential-sensitive mitochondrial dye followed by rapid tissue processing and high-resolution confocal imaging. Image stacks are analyzed to quantify mitochondrial network connectivity, area, and fragmentation. The protocol is first validated in dystrophic and healthy skeletal muscle to confirm expected differences in mitochondrial morphology and is subsequently adapted to visualize and quantify mitochondrial networks in tSCs identified using S100β reporter mice. This approach enables the analysis of mitochondrial morphology within intact neuromuscular tissues without requiring transgenic mitochondrial reporters or specialized imaging platforms. The protocol requires only equipment commonly available in university core facilities and can be adapted to other thin or superficially accessible tissues.