Muscle histology

Isolated muscle was mounted with tragacanth gum (G1128; MilliporeSigma) and quick frozen in liquid nitrogen–cooled 2-methylbutane. Samples were stored at −80°C until sectioning into 12-µm sections. Cryostat sections were processed and fixed in acetone. Hematoxylin and eosin (H&E) staining was conducted, and muscle fiber cross-sections were visualized using EVOS FL Color Imaging system (Thermo Fisher Scientific). Succinate dehydrogenase staining of muscle fiber cross sections were visualized with an Eclipse 80i microscope (Nikon, Tokyo, Japan) with NIS-Elements AR 3.0 software (Nikon). For immunohistochemistry, the sections were fixed with 10 min incubation in ice-cold acetone, then blocked in TSA blocking reagent (FP1012; PerkinElmer, Waltham, MA, USA) for 2 h, and incubated with primary antibodies: TOM20 (sc-11415; Santa Cruz Biotechnology) 1:500, SQSTM1/p62 (H00008878; Novus Biologicals, Littleton, CO, USA) 1:100, LC3 (0231-100; NanoTools Antikoerpertechnik, Teningen, Germany) 1:250, and BNIP3 (ab10433; Abcam, Cambridge, United Kingdom) 1:200; overnight at 4°C, followed by the appropriate secondary antibody. Coverslips were mounted with Mowiol 4–88 (MilliporeSigma) + DAPI and examined with a fluorescence microscope [Nikon 80i upright+ and Scientific EZ monochrome charge-coupled device (CCD) camera (Roper Technologies, Sarasota, FL, USA) with deconvolution software analysis (NIS Elements; Nikon)]. Nonfluorescent images were taken with a 5-megapixel color CCD camera (Nikon).