Histological Analysis of Tibialis Anterior Muscle

TA muscles were embedded in tragacanth gum (Sigma-Aldrich) (5% w/v in water), frozen in liquid nitrogen-cooled isopentane and stored at −80 °C until further processing. Air-dried 10 μm-thick transverse sections were fixed with 4% PFA and stained with hematoxylin and eosin (HE) or for succinate dehydrogenase (SDH) activity using conventional procedures^{16 (link),17 (link)}. Image acquisition was performed with a NanoZoomer 2.0-HT slide scanner equipped with the fluorescence module L11600–21 (Hamamatsu Photonics). Myofiber cross-sectional area (CSA) was analyzed using FIJI image analysis software (ImageJ 1.51s version, available at http://imagej.net/) from fluorescence pictures taken from HE-stained TA sections. CSA (μm²) was determined from at least 300 fibers per muscle. The percentage of myofibers with abnormally positioned nuclei (either centralized or internalized) was calculated from at least 300 fibers per TA using the cell counter plugin in FIJI analysis software. The total number of myofibers in each TA was also counted using FIJI.