Bs 3550r

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).

For immunohistochemical analysis, mice were first anesthetized with ketamine (16 mg/kg body weight) and perfused transcardially with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde (PFA) in PBS. The right hind limbs of the mice were removed from the skin and gastrocnemius muscle and immersed in 4% PFA overnight. The tibia and excess muscles were then removed, and the TA muscles were fixed for 3 h in 4% PFA, then dehydrated with 30% sucrose for 24 h. The TA muscles were then embedded in optimal cutting temperature solution (OCT), frozen, and sectioned at a thickness of 10 µm. The sections were blocked and permeabilized with 10% normal bovine serum and 0.2% 30%Triton X-100 in PBS for 2 h, then incubated overnight at 4 °C with primary antibodies. Subsequently, the sections were washed with PBS and stained with the Alexa-Fluor-488 or Alexa-Fluor-594-labeled goat anti-mouse or rabbit Ig secondary antibodies (diluted 1:500). For section immunofluorescence, the following antibodies were used: TMEM30A (rabbit polyclonal, ab217330, Abcam, USA), Pax7 (rabbit polyclonal, AF7584, Affinity, USA), Laminin (rabbit polyclonal, L9393, Sigma, USA), MYH3 (rabbit polyclonal, 22287-1-AP, Proteintech, USA), MYOD (rabbit polyclonal, 18943-1-AP, Proteintech, USA), and MYOG (rabbit polyclonal, bs-3550R, Bioss, USA).

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.