Frozen GAS muscles were embedded in a mounting medium (Tissue-Tek; Sakura Finitek Europe, Zoeterwoude, The Netherlands) and cut in serial transverse sections (14–16 μm) using a cryostat (Leica CM3050S, Wetzlar, Germany) at −22°C. Sections were mounted on gelatinized slides (0.02%), incubated for 5 min in a fixing buffer (Viscor et al., 1992) in order to prevent shrinkage or wrinkling and finally stained for: (1) myofibrillar adenosine triphosphatase (mATPase), following preincubation in alkaline solution (pH 10.7), to differentiate between slow- and fast-twitch fibers (Brooke and Kaiser, 1970); (2) succinate dehydrogenase (SDH), to identify aerobic and anaerobic fibers (Nachlas et al., 1957); (3) endothelial adenosine triphosphatase (eATPase), to reveal muscle capillaries (Fouces et al., 1993). A representative combined image can be seen in Figure 1. A complete set of representative images for all the histochemical assays from the four experimental groups at the two sampling time points is included as Supplementary Material (Supplementary Figure 1).

Representative microphotographs of gastrocnemius cross-section histochemical assays. (A) Endothelial ATPase stain (arrows indicate muscle capillaries); (B) myosin ATPase stain; (C) succinate dehydrogenase stain. () Slow oxidative fiber (SO); () fast oxidative glycolytic fiber (FOG); (+) fast intermediate glycolytic fiber (FIG); # fast glycolytic fiber (FG). Bar represents 100 μm.

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