Fresh muscle tissues were mounted in frozen section medium (Thermo Fisher Scientific, cat#6520) and sliced by cryostat (Leica, cat#CM1860) to obtain 10 µm cryosections.
The cryosections or cultured cells were fixed in PBS containing 4% paraformaldehyde (Sigma-Aldrich, cat#30525) for 15 min, permeabilized in 0.5% Triton X-100 for 15 min at room temperature, blocked in PBS containing 1% BSA (Beyotime Biotechnology, cat#ST023). Anti-PAX7 (Developmental Studies Hybridoma Bank, 1:100), anti-Laminin (Abcam, cat#ab11575, 1:500) or anti-MyHC (Millipore, cat#05-716, 1:1000) was applied and incubated overnight at 4 °C. Alexa 488- or Alexa 594-labeled anti-mouse or anti-rabbit secondary antibodies (Invitrogen, 1:1000) were applied to incubate for 1 h at room temperature. The samples were then stained with 1 μg/mL DAPI (Vector Laboratories, cat#H-1200) and finally mounted with antifade mounting media (Vector Laboratories, cat#H-100) for subsequent imaging.
The cryosections or cultured cells were fixed in PBS containing 4% paraformaldehyde (Sigma-Aldrich, cat#30525) for 15 min, permeabilized in 0.5% Triton X-100 for 15 min at room temperature, blocked in PBS containing 1% BSA (Beyotime Biotechnology, cat#ST023). Anti-PAX7 (Developmental Studies Hybridoma Bank, 1:100), anti-Laminin (Abcam, cat#ab11575, 1:500) or anti-MyHC (Millipore, cat#05-716, 1:1000) was applied and incubated overnight at 4 °C. Alexa 488- or Alexa 594-labeled anti-mouse or anti-rabbit secondary antibodies (Invitrogen, 1:1000) were applied to incubate for 1 h at room temperature. The samples were then stained with 1 μg/mL DAPI (Vector Laboratories, cat#H-1200) and finally mounted with antifade mounting media (Vector Laboratories, cat#H-100) for subsequent imaging.