Hematoxylin-eosin (H&E) and immunofluorescence staining

A detailed protocol for muscle histological characterization using H&E staining and myofiber typing using immunofluorescence are published in Bio-Protocol (Wang et al., 2017). Briefly, fresh TA muscles were embedded in OCT compound, and frozen in isopentane chilled on dry ice. Then the tissues were cut at 10 µm thickness by Leica CM1850 cryostat. Muscles sections were fixed with fresh 4% PFA for 10 min, washed in 100 mM glycine three times each 10 min, followed by PBS wash for three times and blocking for 1 hr at room temperature. The primary antibody was incubated at 4°C for overnight. Fluorescence secondary antibody was diluted and incubated at room temperature for 1 hr. Images were taken with a Leica DM6000 microscope with a 20× objective. H&E staining images were captured with a Nikon D90 digital camera mounted on a microscope.