Background

Skeletal muscle is a highly organized tissue composed of several distinct compartments of muscle fibers. Each muscle fiber is a multinucleated single cell, enclosed by sarcolemma, and is predominantly made up of myofibrils (Seeley et al., 2007). Muscle fibers that have low contraction speed are called slow twitch or type I fibers. Slow twitch fibers have high mitochondrial density and express myosin isoform Myh7 (Lompre et al., 1984; Talmadge and Roy, 1993). Type II fibers are divided into type IIa and type IIb fibers. Both of these fibers have high contraction speed, thus generating greater force compared to type I fibers. Type IIa fibers have moderately high contraction speed and are anaerobic in nature. Type IIa fibers also have high density of mitochondria with increased oxidative potential. Type IIa fibers predominantly contain myosin isoform Myh2 in mammals. Type IIb fibers have very high contraction speed and are anaerobic in nature. Type IIb fibers have low mitochondrial density and predominantly contain myosin isoform Myh4. Muscle fibers that are neither Type IIa nor Type IIb fibers are called Type IIx or Type IId, and have high expression in the diaphragm and hence is omitted from this study (Schiaffino and Reggiani, 1994; Westerblad et al., 2010). Mouse tibialis anterior, a hind limb skeletal muscle made of Type I, Type IIa and Type IIb myofibers serves as a suitable muscle to study the robustness of the immunohistochemical staining procedure.

Other methodologies to distinguish the different muscle fiber types are myosin ATPase histochemical staining of frozen muscle sections pre-incubated in alkaline pH showing light staining for Type I fibers and dark staining for Type II fibers. Muscle sections pre-incubated in acidic pH showed dark ATPase staining for Type I fibers, light staining for Type IIa muscle fibers and intermediate staining for Type IIb fibers (Brooke and Kaiser, 1970, Guth and Samaha 1970). Quantitative estimation of mitochondrial enzymes like glycerophosphate oxidase (GP-OX) and succinate dehydrogenase (SDH) have been used to distinguish Type IIa and Type IIb fibers in mouse and rabbit tibialis anterior muscle but gave variable ratios of enzyme activities in mice and rabbit muscles (Reichmann and Pette, 1984). There have been quantification methodologies of specific metabolic enzymes like lactate dehydrogenase and malate dehydrogenase on frozen serial sections of muscles (Hintz et al., 1984). However, these protocols are extensive and may give non-specific signals. The protocol detailed here uses specific antibodies that recognize different Myosin Heavy chain isoforms on serially cut frozen tibialis anterior mid-belly region sections that is readily detected with a suitable secondary antibody. After image acquisition, the number of each fiber type is quantified using ImageJ software.