Myotube diameter measurement and quantification

The diameter of myotubes was analyzed by MHC staining. Briefly, C2C12 cells were seeded and differentiated on the glass coverslips. Then C2C12 myotubes were fixed and incubated with mouse anti-MHC (1:3,000) (R&D Systems, USA) for 1 hour. When the antibody incubation fluid was removed, the slips were incubated with affinity-purified Alexa Fluor dye-conjugated goat anti-mouse antibody (1:100) (Life Technologies, USA) for 30 min. The slips were photographed with the fluorescence microscopy. The diameter of myotubes was evaluated by the Image J software (27).