Image analysis system software

Soleus histological sections (12 µm thick) from CC (n = 6) and CT (n = 6) were obtained in a cryostat JUNG CM1800 (Leica, Germany) at −24 °C to determine muscle fiber-type frequency and cross-sectional area (CSA), using myofibrillar adenosine triphosphatase (m-ATPase) histochemistry after pre-incubation at pH 4.35. Muscle fiber types were classified as Types I, Ic/IIc, and IIa. Fiber CSA for each fiber type, and fiber-type frequencies were determined using Image Analysis System Software (Leica, Germany). At least 200 fibers at different points of soleus muscle were measured and their frequency was expressed as the number of fibers per type against the total number of fibers measured.

Collagen content in soleus muscle was determined using Picrosirius red staining. Briefly, transverse cryosections (12 μm thick) of all samples were placed on the same slide to minimize staining differences; sections were incubated with saturated picric acid solution followed by Picrosirius red (0.1% Sirius red in saturated picric acid) for 3 min., dehydrated and mounted in Permount. Eight color pictures per sample were captured using the microscope with polarized light (400X magnification). Light intensity and filters alignment parameters used were the same for all samples. Quantitative analysis of endomysium collagen type I staining intensity was determined by measuring the grayscale with the Image Analysis System Software (Leica, Germany). The gray values were normalized by mean fiber area.