Cross-sectional area of muscle fibers

The MA, TA and SOL, each excised as a whole muscle, were embedded in Tissue-Tek OCT compound (Sakura Finetec, Torrance, CA, USA) in a slightly stretched state so as to maintain a length close to the resting length (L0), and stored at − 80 °C until sectioning, as reported [12]. Cross sections (10 μm) were cut from the middle portion of each muscle with a cryostat (CM1900, Leica Microsystems, Nussloch, Germany) at − 20 °C. The sections were air-dried and fixed with 4% paraformaldehyde in 0.1 M PBS (pH 7.5). The sections were then stained with hematoxylin and eosin (HE) and observed under a light microscope (BX61, Olympus Co., Tokyo, Japan). Micrographs were taken with a digital camera (DP-72, Olympus Co.) connected to a personal computer. The cross-sectional size of muscle fibers was evaluated by measuring the cross-sectional area (CSA) [13, 14]. The CSAs of 100 muscle fibers in the superficial portion were measured with image analysis software (Image J 1.45) and averaged to obtain the mean values in each mouse.