Protocol for muscle fiber type and cross-sectional area analysis in cryosections of whole lower mouse hindlimbs

We outline a protocol to visualize all mouse lower hindlimb skeletal muscles simultaneously. We describe procedures for orientating the whole lower hindlimb in gum tragacanth prior to freezing, simplifying the proceeding experimental steps, and enhancing the comprehensiveness of characterizations. We then detail steps for quantifying muscle fiber size and fiber type characteristics in a single cryosection using histochemistry and immunofluorescence. This protocol can be applied to histological and (immuno)histochemical evaluations such as muscle regeneration, fibrosis, enzymatic activity, and glycogen content. • Orientate mouse lower hindlimb in tragacanth to enhance clarity and simplify steps • Simultaneously visualize myofibers in different muscles in a single cryosection • Analyze myofiber size and types with immunofluorescence • Applicable for evaluating muscle degeneration, fibrosis, and glycogen content Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. We outline a protocol to visualize all mouse lower hindlimb skeletal muscles simultaneously. We describe procedures for orientating the whole lower hindlimb in gum tragacanth prior to freezing, simplifying the proceeding experimental steps, and enhancing the comprehensiveness of characterizations. We then detail steps for quantifying muscle fiber size and fiber type characteristics in a single cryosection using histochemistry and immunofluorescence. This protocol can be applied to histological and (immuno)histochemical evaluations such as muscle regeneration, fibrosis, enzymatic activity, and glycogen content.