Anti myosin heavy chain mhc

Procedures for determining the fiber cross-sectional area (CSA) were conducted as previously described [22 (link)]. 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). The CSA and number of myofibers with central nuclei among the individual myofibers and the diameter of the myotubes were determined by using Fiji software [23 (link)].
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) (R&D Systems, Minneapolis, MN, USA) (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). The fluorescence score was calculated by multiplying the average light intensity and area of fluorescence staining with Fiji software.