Lipid droplet staining

Muscles (> 3 sections per muscle) were sectioned in the mid-belly at a thickness of 10 μm and were stained with BODIPY-493/503 (Invitrogen, Carlsbad, CA) at 1:200 dilution for 1 hour to identify neutral lipid in muscle (N = 6 per group). Sections were mounted in Vectashield. Sections were visualized using a confocal microscope (Zeiss 510), and fluorescence of ~600 muscle fibers per group was quantified using ImageJ software (NIH, Bethesda, MD) as previously described [48]. Briefly, the integrated density, mean gray value, and area were measured for individual muscle fibers (~100 myofibers per animal), along with several background readings. The fluorescence for each muscle fiber was calculated by the following equation: Integrated density – (area of muscle fiber × mean fluorescence of background readings) × 100.