Muscle fiber cross-sectional area

For cross-sectional area (CSA) calculations, 5 μm thick transverse sections were obtained from the mid-belly area of right EDL muscles. After toluidine blue staining, they were randomly selected and photographed using light microscopy (Zeiss Axioskop) with a digital camera (Hamamatsu CCD). Myofiber CSA was measured by tracing the external border of individual myofibers using Image J software. 20 microscopic fields per muscle were analyzed at 20x magnification. Cross-sectional areas were determined for at least 300 muscle fibers per animal. Myofibers exhibiting evidence of tears or processing artifacts were excluded from the analysis.