Myhc iib efluor 660

For immunostaining, cells grown on plates were fixed with 4% paraformaldehyde at room temperature for 15 min and subsequently permeabilized with 0.3% Triton X-100/ PBS. Cells were blocked in 10% goat serum diluted in 0.1% Triton X-100/ PBS at room temperature for 1 h. Primary antibodies were diluted in 1% goat serum/ 0.1% Triton X-100 and incubated at room temperature for 2 h. Cells were washed and secondary antibodies were diluted in 1% goat serum/0.1% Triton X-100 and incubated at room temperature for 1 h. Cells were stained with Hoechst dye (1:2,000 in PBS) at room temperature for 10 min. Primary antibodies include: Pax3 (DSHB, 1:20), Pax7, (DSHB, 1:20), MyoD (Santa Cruz Biotechnology, sc-377460, 1:100), MyoG (Santa Cruz Biotechnology, sc-12732, 1:100), MYH1 (clone MF20, DSHB, 5μg/ml). MYHC-IIb eFluor 660 (50-6503-32; Thermo Fisher; 1:100), α-actinin (sc-7453; Santa Cruz; 1:500), 8-oxo-guanine (ab206461; Abcam; 1:400). Fusion index was calculated as a ratio of the number of tdTO+ or MuSC nuclei within a multinucleated myotube to the total number of tdTO+ or MuSC nuclei. A minimum of 4 independent microscopic fields was used for each group over three independent differentiation experiments at 24 and 36 hours in DM.