Anti mhc antibody

Myotubes were fixed in PBS with 4% paraformaldehyde for 15 min at room temperature and then permeabilized in PBS containing 0.2% Triton X-100 for 5 min at room temperature. The myotubes were then blocked with PBS containing 5% goat serum and 2% BSA for 1 h at room temperature and then incubated overnight at 4°C with anti-MHC antibody (Abcam; Cambridge, UK; ab91506, 1:200). Next, the myotubes were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Abcam; ab150077, 1:500) for 1 h at room temperature and then mounted with Fluoroshield Mounting Medium containing DAPI (Abcam; ab104139). Images were acquired with a fluorescence microscope (Nikon; Tokyo, Japan) and analyzed with Image J software (version 1.46r, NIH, USA).
Myotube diameters were measured as previously described [38 (link)]. Briefly, 20 pictures were taken per well, and the diameters of the five largest myotubes (those containing ≥ 3 nuclei when viewed at ×100 magnification) in each picture were measured. Next, the mean diameter of a single myotube was calculated based on three independent measurements. The measurement points were separated by 200 μm. This method was also used to calculate the mean diameter ± SD of the 100 largest myotubes in each well.

Immunocytochemistry cellular immunofluorescence was conducted as previously described [41 (link)]. The MDSCs were first fixed in 4% PFA (Sigma) and permeabilized with 1% Triton X-100 (Invitrogen). The cells were incubated with an optimal concentration of anti-MHC antibody, anti-CD146 antibody, anti-PAX7 antibody, and anti-PW1 antibody (1:200, all from Abcam) overnight at 4 °C, incubated with an Alexa Fluor 488 or 546 secondary antibody (1:800, Invitrogen), followed by rinsing three times with PBS. Then, cell nuclei were stained with Dapi (Invitrogen) prior to being imaged by fluorescent microscope (Olympus). The number, length, diameter, and maturation index data of myotubes were obtained from at least three images of each sample using ImageJ software, and cell positive rate of each marker was calculated by normalizing the positive cell number to the total cell number.