paraformaldehyde at ambient temperature for 20 min, washed with phosphate
buffered saline (PBS), permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) at
ambient temperature for 30 min, and washed with PBS again. The polyclonal
anti-myogenic differentiation 1 (MYOD1) (BS-2442R, Bioss, Beijing, China),
anti-paired box 7 (Pax7) (AB-528428, Abcam, Cambridge, UK), and anti-desmin Po
(BS-20702R, Bioss) were diluted in PBS with 5% bovine serum albumin (BSA) at a
ratio of 1:100 and incubated at ambient temperature for 2 h. The horseradish
peroxidase (HRP)-labeled Goat Anti-Mouse Immunoglobulin G (IgG) (H + L) (A0216,
Beyotime, Shanghai, China) or Goat Anti-Rabbit IgG-HRP antibody (BS-3550R,
Bioss) was diluted in PBS with 5% BSA at a ratio of 1:200 and incubated for 2 h
at ambient temperature in the dark. Finally, the BSC was incubated in the 0.1
μg/mL 4’,6-diamidino-2-phenylindole, dihydrochloride solution for
30 min in the dark and observed with a fluorescence microscope (WF10X, Olympus,
Tokyo, Japan).