Muscle fiber cross-sectional area

To determine muscle fiber size via cross-sectional area (CSA), freshly isolated gastrocnemius samples were mounted with Optimal Cutting Temperature (OCT) compound, and snap-frozen with liquid nitrogen-cooled isopentane. Mounted samples were sectioned using a cryostat (Leica; Wetzlar, Hesse, Germany). Three serial sections (10 µm) spanning the length of each gastrocnemius muscle were prepared and stained with hematoxylin and eosin (H&E). Images were acquired using an Olympus IX71 microscope and cellSens Standard software (Center Valley, PA, USA). Muscle fiber CSA was quantified by fiber diameter (ImageJ; National Institutes of Health, Bethesda, MD, USA). An evaluator manually outlined and quantified individual fibers for a separate blinded evaluator, who then grouped results accordingly for data analysis. Results from each of the 3 sections per muscle were averaged prior to statistical analysis.