Dapi pi

Paraffin-embedded gastrocnemius and TA were cross-sectionally cut into 4-μm sections as above. After antigen retrieval, permeabilization, and goat serum blocking, primary antibody incubation was conducted overnight at 4 °C; primary antibodies included: mouse anti-MHC (1:1000), rabbit anti-TGF-β1 (1:100), rabbit anti-LC3B (1:200), rabbit anti-wheat germ agglutinin (WGA; 1:50; Abcam, USA) and rabbit anti-HMGB1 (1:400). Samples were then stained for 1 h with secondary Alexa Fluor 488- or Alexa Fluor 594-conjugated anti-mouse or anti-rabbit secondary antibodies (1:300, Invitrogen, USA), followed by a 5 min DAPI/PI (Sigma, USA) staining. Samples were then imaged using a fluorescence microscope. Similar procedures were carried out for C2C12 myotubes.

Paraffin-embedded GAS underwent cross-sectional cuts, which were made into 4-μm sections as previously stated [16 (link)]. Following antigen retrieval, permeabilization, and goat serum blocking, primary antibody (mouse anti-slow MHC antibody and rabbit-anti-fast MHC antibody; Abcam) incubation was implemented at 4 °C. Next, samples were stained for 1 h using secondary Alexa Fluor 488-conjugated goat anti-mouse and Alexa Fluor 647-conjugated goat anti-rabbit IgG antibodies (Abcam, ab150113 and ab150079), followed by 5 min DAPI/PI (Sigma, USA, D9542) staining. The samples were then imaged with a fluorescence microscope. Similar processes were oriented with C2C12 cells.