Mouse anti desmin clone d 33

Cultured SC were detached at day 4 and day 8 after isolation by using HyClone HyQTase, and the reaction was stopped by adding PBS containing 10% FBS. After centrifugation (10 min, 300 g, 10 °C), cells were re-suspended in 1 mM EDTA in PBS and fixed in 4% paraformaldehyde (PFA). Following fixation, cells were permeabilized with 0.1% (Desmin, MyoG) or 0.5% (Pax7, MyoD1) Triton X-100 in PBS, blocked with normal rabbit serum, and incubated overnight with the respective primary antibody: mouse anti-Desmin (clone D-33, DAKO, 1:80), mouse anti-Pax7 (Developmental Studies Hydridoma Bank, 1:50), mouse anti-MyoG (clone F5D, abcam, 1:50), or mouse anti-MyoD1 (clone 5.2F, abcam, 1:200). A portion of each sample was incubated with normal rabbit serum instead of primary antibody as a negative control. After two additional washing steps, samples were incubated with a rabbit anti-mouse Alexa488 antibody (Thermo Fisher Scientific, 1:1000) for 1 h at room temperature and washed again. In total, 5000 events per sample were analyzed by using an argon-equipped Gallios Flow Cytometer (Beckman Coulter) and evaluated with Kaluza software (Beckman Coulter).

The fixed and differentiated cells were permeabilized with 0.1% (Desmin) or 0.3% (MHC) Triton X-100, blocked with normal rabbit serum, and incubated with the primary antibody, namely mouse anti-Desmin (clone D-33, DAKO, 1:80) or mouse anti-skeletal fast Myosin (MHC; clone MY-32, Sigma Aldrich, 1:400), overnight. After being washed with PBS, the samples were incubated with a rabbit anti-mouse Alexa488 antibody (Thermo Fisher Scientific, 1:1000) for 1 h at room temperature, and subsequently, cell nuclei were stained with DAPI (15 min, 1 μg/ml). For fluorescence microscopy, the Nikon Diaphot 300 microscope and Olympus cell^F software were used. Micrographs were merged by using Adobe Photoshop CC 2017; contrast and brightness were adjusted to the same degree in every sample group.
MHC+ myotubes containing ≥2 nuclei were encircled to determine the myotube area by using ImageJ 1.49v. For each experiment, 10–12 random sections per population were analyzed. Micrographs of the Desmin-stained cells were used to determine cell fusion rate. The number of nuclei in the fused multinucleated cells (≥ 2 nuclei) was divided by the total number of visible nuclei in 5 random sections for each experiment and subpopulation. Experiments were repeated independently three times with cells obtained from three animals.