Mouse anti myosin

Immunofluorescence staining of skeletal muscle sections was performed as previously described (Wu et al., 2012b; Yang et al., 2019a). Briefly, the frozen skeletal muscle sections were washed for 10 minutes with 0.5% phosphate-buffered saline/Triton X-100 (PBST) (3×), and then blocked with 5% normal goat serum in PBST for 1 hour at room temperature. The sections were thereafter incubated with primary antibodies in PBST at 4°C overnight. The following antibodies were used: mouse anti-myosin (skeletal, fast) antibody (1:500; Cat# M4276; Sigma-Aldrich) and mouse anti-myosin (skeletal, slow) antibody (1:500; Cat# M8421; Sigma-Aldrich). Then, the sections were washed 10 minutes with PBST (3×), and then incubated with Alexa Fluor 568-conjugated goat anti-mouse IgG (1:500; Cat# 20100; Biotium, Fremont, CA, USA) or Alexa Fluor 488-conjugated goat anti-mouse IgG (1:500; Cat# 20010; Biotium) for 1 hour at room temperature. The sections were thereafter washed with PBST (3×). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (Cat# ZLI-9556; ZSGB-BIO) and mounted onto glass slides with anti-fade solution. All images were captured on a Olympus FV-1200 confocal microscope (Olympus, Center Valley, PA, USA) and analyzed with Imaris 9.0.1 software (Abingdon, Oxfordshire, UK).