Immunofluorescence and Quantification of Myotubes

The myotube diameter was detected using MHC staining. Briefly, C2C12 cells were grown and differentiated into myotubes. Then the C2C12 myotubes were fixed in 4% paraformaldehyde, permeabilized in 0.1% saponin, blocked in 1% BSA, and then incubated overnight at 4°C with mouse anti-MHC antibody (1:200; R&D Systems). Myotubes were then rinsed with phosphate-buffered saline (PBS) and incubated for 30 min with 1:100 affinity-purified Alexa Fluor dye-conjugated goat anti-mouse antibody (Life Technologies, Carlsbad, CA, USA) at room temperature. Immunostained myotubes were visualized under a fluorescence microscope (Zeiss, Germany). To estimate myotube size, myotubes were defined as all multinucleated cells positive for the MHC stain and containing at least three nuclei. The diameter of at least 100 myotubes per condition was measured using ImageJ software (NIH, Bethesda, MD, USA), and the average diameter per myotube was calculated as the mean of three measurements taken along the length of the myotube (Abrigo et al., 2016).