Histological analysis and immunofluorescence
Procedures for the determination of the fiber cross-sectional area (CSA)
were conducted as previously described (Park et al., 2013). Briefly,
fixed transverse sections (7 μm) were cut from the mid-belly of the
gastrocnemius with a cryostat at -20°C and then stained with hematoxylin
and eosin (H&E). Images were obtained at 40× magnification and
quantified using ImageJ software (National Institutes of Health,
Bethesda, MD). The CSA and number of myofibers with central nuclei among
the individual myofibers were determined by using ImageJ 1.48 software
in five random fields for each section.
For immunofluorescence analysis, cells were seeded onto sterile
preprocessed glass coverslips that were precoated with 1% gelatin.
After C2C12 myoblasts had differentiated into myotubes, the cells were
washed twice with PBS, followed by fixation in 4% paraformaldehyde for
15 min. After being rehydrated in PBS, the cells were blocked for 30 min
in 1% bovine serum albumin (BSA) in PBS containing 0.2% Triton-X
(PBST). Then, the cells were incubated with an anti-myosin heavy chain
(MHC) (#MAB4470, R&D Systems,MN) (1:100) in 1% BSA/PBST overnight at
4°C. Cells were then incubated with a fluorescence-labeled secondary
anti-mouse antibody (1:1000) and DAPI (1:1000) at room temperature for 1
h. The specimens were examined under an FV10i laser scanning confocal
microscope (Olympus, Tokyo, Japan).