Assessment of haematoxylin–eosin staining

Myotube diameter of H&E stained C2C12 myotubes was quantified on images using Image J software as described in our previous report. 17 Briefly, 10 fields were randomly selected to determine the average diameters of at least 100 myotubules for each condition. The average diameter per myotube was calculated as the mean of three measurements taken along the long axis of the myotube. Results of three independent experiments were used for statistical analysis. Cross‐sectional area of myofibres and adipocytes were quantified on H&E stained tissue sections by using Image J software. Briefly, 10 images were randomly taken for each individual muscle and adipose tissue sample, and the cross‐sectional area of all myofibres and adipocytes on the image were measured. Results of the mice in the treatment groups were used for statistical analysis.