Abstract
Mammalian skeletal muscle is a metabolically active tissue that is made up of different types of muscle fibers. These myofibers are made up of important contractile proteins that provide force during contraction of the muscle like actin and myosin. Murine myofibers have been classified into 4 types: Type I, Type IIa, Type IIb and Type IIX. Each muscle fiber has been identified with specific type of MyHC expressed, which in turn gives differential contractility to the muscle. There have been well-known methodologies to identify different myofibers: histochemical myosin ATPase staining which uses the differential ATPase activity between slow and fast fibers, quantification of metabolic enzymes like malate dehydrogenase and lactate dehydrogenase on specific fragments of muscle fibers. The drawback of these techniques is that they cannot differentiate the subtypes of myofibers, for example, Type IIa and Type IIb. They should be used in conjunction with other known histochemical staining techniques. Here, we devise a direct and robust immunohistochemical staining methodology that utilizes the differential expression of MyHC isoforms in different myofibers types, thus efficiently distinguishing the heterogeneity of the muscle fibers. We use antibodies that specifically recognize Type I, Type IIa and Type IIb fibers on serially cut frozen mouse tibialis anterior sections that can be quantified by ImageJ software.