The histological analysis was performed as described previously (Zhang et al. 2019 (link)). The cross-sectional area and minimal Feret’s diameter of the gastrocnemius muscles were determined by immunostaining with an anti-laminin antibody (dilution: 1:200; Abcam, Cambridge, MA, USA) (Moresi et al. 2010 (link)). The primary antibody was detected by Alexa Fluor-594 (dilution: 1:1000; Life Technologies, Carlsbad, CA, USA) fluorescent dye conjugated to an anti-rabbit secondary antibody. Myotubes were fixed with 4% paraformaldehyde for 10 min, blocked with 5% goat serum for 30 min, and incubated with anti-myosin (dilution: 1:500; ZSGB-BIO, China) at 4 °C overnight. The secondary antibody was goat anti-mouse IgG H&L (Alexa Fluor® 488) (dilution: 1:1000; Life Technologies, Carlsbad, CA, USA). Nuclei were stained using Vectashield with DAPI (Vector, USA). Images were visualized through a Leica TCS SPS III confocal microscope (Leica, Germany). The diameter of each individual myotube was measured and the average overall diameter was calculated. Images were analyzed using Image-Pro Plus 6.0.
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