Quantifying Muscle Fiber Morphology

Determination of the cross-sectional area (CSA) and the percentage of slow and fast muscle fibers can be found in our previous reports [44 (link),50 (link)]. In brief, the soleus muscle sections were prepared with a Leica CM 1900 cryostat (Leica, Braunschweig) at −20 °C. Sections were incubated with primary antibodies MyHC I(β) slow (1:100 Sigma, St. Louis, MO, USA), MyHC fast (1:60, DSMZ) for 1 h at 37 °C and secondary antibodies Alexa Fluor 546 (1:1000; Molecular Probes, Waltham, MA, USA) for 60 min in the dark at room temperature. The soleus muscle sections were photographed with a Leica Q500MC fluorescent microscope at magnification ×20. Image analysis was processed by the ImageJ 1.52a software. At least 150 fibers were analyzed in each muscle sample (n = 8) for myofiber CSA measures, and at least 10 cross-sections per sample were examined to determine the percentage of different muscle fiber types in the sample (n = 8).