Primary anti desmin

Immunofluorescence was used to identify the isolated skeletal muscle satellite cells. Briefly, cells grown in 12-well plate were firstly washed with PBS for three times. and fixed with 4% paraformaldehyde for 15 min. After being washed three times with PBS, the cells were permeabilized with 0.25% Triton X-100 per well for 10 min and blocked at 4°C overnight. Afterwards, cells were incubated with 1:100 diluted primary anti-Desmin (Abcam, China) or anti-Pax7 (Abcam, China) for 1 hour at room temperature. After washing, 1:2,000 diluted fluorescent secondary antibody (Thermo Fisher, China) was used to incubate the cells for 1 hour at room temperature. The cells were then washed, added with 4′,6-diamidino-2- phenylindole (Invitrogen, Carlsbad, CA, USA) and incubated for 15 min at room temperature to stain the cell nuclei. In the end, samples were captured using a fluorescence microscope (OLYMPUS, Tokyo, Japan).

The isolated skeletal muscle satellite cells at 70%-80% fusion and the cells that were induced to differentiate after transfection were used for immunofluorescence detection following the method of Luo et al. [32 (link)]. Briefly, cells grown in 12-well plates were washed three times with precooled PBS and fixed with 4% paraformaldehyde for 15 min. Thereafter, the cells were permeabilized with 0.25% Triton X-100 per well for 10 min and blocked at 4°C overnight. Afterwards, cells were incubated with 1 : 100 diluted primary anti-Desmin (Abcam, China), anti-Pax7 (Abcam, China), or anti-MyHC (Santa, USA) antibodies for 1 hour at room temperature. Then, 1 : 100 diluted fluorescent secondary antibody (Thermo Fisher, China) was incubated with the cells for 1 hour at room temperature. After being washed by PBS three times, DAPI (Invitrogen, USA) was added to the cells and then incubated for 15 min at room temperature to stain the cell nuclei. Lastly, samples were captured using a fluorescence microscope (OLYMPUS, Japan).