Muscle histology

One GA muscle from each mouse was dissected, placed in optimal cutting temperature compound, rapidly frozen in isopentane cooled in liquid nitrogen, and stored in a −80°C freezer. Frozen cross sections were cut from the midbelly of each muscle at a thickness of 10 mm using a cryostat (Leica, Germany) and placed on a glass slide. Sections were incubated with hematoxylin solution for 2 min, washed in deionized water, and then incubated with eosin solution for 2 min. Sections were then washed in deionized water, dehydrated in ethanol, and then mounted. The sections were observed using an inverted microscope, and images were captured. Photoshop was used to calculate the pixel count of the cross section of the muscle fiber, and this value was converted to an area. Muscle fiber cross‐sectional area (CSA) was measured in 100 randomly selected fibers per mouse.