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

Abstract

We outline a robust, simple, and cost-effective method to simultaneously visualize all mouse lower hindlimb skeletal muscles. We describe procedures for orientating the whole lower hindlimb in gum tragacanth prior to freezing, simplifying the proceeding experimental steps, and enhancing the clarity and comprehensiveness of characterizations. We then detail steps for quantifying muscle fiber size and fiber type characteristics in a single cryosection using immunofluorescence and histochemistry. This protocol can be applicable for commonly used histological and (immuno) histochemical evaluations such muscle degeneration/regeneration, fibrosis, immune cell infiltration, enzymatic activity and glycogen content. HighlightsO_LIEmbedding and orientating mouse lower hindlimb in tragacanth before freezing enhances clarity and simplifies steps. C_LIO_LISimultaneously visualizing myofibers in different muscle compartments enables efficient and comprehensive evaluations of morphology and physiological characteristics. C_LIO_LISingle cryosection analysis of myofiber size and types with immunofluorescence. C_LIO_LIApplicable for evaluating muscle degeneration, fibrosis, and immune infiltration. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/608909v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@9d3fe4org.highwire.dtl.DTLVardef@1cdcc85org.highwire.dtl.DTLVardef@2e494eorg.highwire.dtl.DTLVardef@cebfc6_HPS_FORMAT_FIGEXP M_FIG C_FIG