Mab4470

Cells were seeded on gelatin and fibronectin-coated glass coverslips at 40% confluency. Following overnight culture, the growth media containing 10% FBS were replaced with differentiation media containing 2% horse serum. At the indicated times, the cells were fixed with 4% paraformaldehyde. The coverslips were washed three times in buffer (PBS + 0.3% Triton) and then blocked using 10% horse serum (Invitrogen). Coverslips were incubated with primary antibody, mouse anti-MHC (Novus Biologicals; MAB4470), diluted in PBS supplemented with 10% horse serum for 24 h. After washing with PBS, coverslips were incubated with an AlexaFluor 488®-conjugated donkey anti-mouse secondary antibody (1:1000, Jackson ImmunoResearch Laboratories) overnight. Fluorescence microscopy was performed using a Z1 AxioObserver inverted fluorescence microscope equipped with an AxioVision MRm camera (Zeiss) operated by ZenPro software (Zeiss). All images were taken using a 20X objective. Myotube density was quantified using morphometric analysis in five randomly selected fields of view from three biological replicates. MHC-positive myotubes were manually traced, and the myotube density was calculated by dividing the area occupied by MHC-positive myotubes by the total area of the field of view.