Muscle fiber-type analysis

GC muscles were harvested from C57BL/6J mice, embedded in the Tissue-Teck O.C.T compound (#4583, Sakura), and rapidly frozen in isopentane fully cooled by liquid nitrogen. GC muscle cryosections (5 μm-thick slices) were blocked in PBS containing 0.3% Triton X-100 and M.O.M blocking reagent (Vector Laboratories, USA) for 1 h at room temperature. Slices were then incubated overnight at 4 °C with primary antibodies, such as the anti-laminin antibody (1:500), and anti-skeletal fast (1:500) or slow myosin (1:1,000) antibodies in the blocking solution with M.O.M. protein concentrate (Vector Laboratories) as previously described⁸¹. Section slices were incubated with secondary antibodies, such as Alexa Fluor 488 anti-mouse IgG antibody (1:2,000) and TRITC-conjugated anti-rabbit IgG antibody (1:40) in PBS containing 0.3% Triton-X-100 for 1 h at room temperature. Image acquisition was performed with an inverted fluorescence microscope, BZ-X710 (Keyence, USA). The fiber cross-sectional area was measured for approximately 200 adjacent muscle fibers in each section for each mouse using ImageJ software (NIH, Bethesda, USA) as described elsewhere⁸².