Ab 528428

BSC were grown on 14 mm coverslip-bottomed dishes and fixed with 4%
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).

To verify whether the cells isolated were BSC, the primary cells were stained for
immu-nofluorescence. Cultured satellite cells were fixed with ice-cold 4%
paraformaldehyde for 20 min, rinsed twice with PBS, and permeated with 0.2%
Triton x-100 (in PBS) at room temperature for 30 min. The permeabilized cells
were blocked with PBS containing 5% bovine serum albumin (BSA) and shaken at
room temperature for 1 h, and incubated with primary antibodies: polyclonal
anti-myogenic differentiation 1 (MYOD1; BS-2442R, Bioss, Woburn, MA, USA),
anti-Pax7 (AB-528428, Abcam, Cambridge, UK), and anti-desmin Po (BS-20702R,
Bioss) in 5% BSA at 1:100 dilutions for 2 h at room temperature. After washing
with PBS, the cells were incubated with HRP-labeled Goat Anti-Mouse IgG (H + L)
(A0216, Beyotime, Shanghai, China) or Goat Anti-rabbit IgG-HRP antibody
(BS-3550R, Bioss) for 2 h at room temperature in dark room and mounted in 0.1
μg/mL DAPI solution for 30 min. The staining of the cells was observed
using a fluorescence microscope (WF10X, Olympus, Tokyo, Japan) afterwards.