Anti slow myhc

Cells were fixed with 4 % paraformaldehyde for 20 min and then permeabilized with 0.3 % Triton-X for 20 min at room temperature. Cells were washed for 5 min with PBST three times after fixing and permeabilization. Cells were incubated in blocking media (PBS-T, 3 % bovine serum albumin, 5 % normal goat serum, 8 % protein concentrate (vector labs), and 0.2 % Tween-20) for 2 h at room temperature. Cells were incubated with primary antibody solution in blocking media with antibodies overnight at 4 °C at the following dilutions: 1:200 anti-fast MyHC (Sigma M4276) and 1:100 anti-slow MyHC (Sigma M8421), anti-Pax7 (DSHB, 1/15), anti-MyoD (1/20, sc-760, clone M-318), and anti-Myogenin (1/50, BD Biosciences-556358). After thorough washing (three times, 15 min each in PBST), the secondary antibody solution containing 1:250 goat anti-mouse 488 (Lifetech) or 1:250 goat anti-rabbit 488 (Lifetech) and Hoechst 33342 (Thermofisher) in blocking buffer was incubated for 1 h at room temperature (dilutions used for all immunofluorescent antibodies had previously been titrated in C2C12 differentiated myotubes). Images of sorted cells were obtained using an Axio-Observer inverted light microscope and AxioVision software (Zeiss, 2015). A merged multidimensional acquisition was set up using a 365-nm reflector for Hoechst and a 470-nm reflector for MyHC, Pax7, MyoD, and MyoG.

Western blotting was performed as previously reported [22 ]. LD muscle samples were homogenised in RIPA lysis buffer (Beyotime biotechnology, Shanghai, China) containing a protease inhibitor (Roche, Shanghai, China). Proteins were separated on 10% SDS–PAGE gel and then were transferred onto a PVDF membrane (Bio-Rad, Shanghai, China). The membrane was blocked with 5% skimmed milk for 1 h at room temperature, and then incubated with the respective primary antibody overnight at 4 °C. Anti-slow MyHC (Sigma, Cat. No. M8421), anti-fast MyHC (Sigma, Cat. No. M4276), PGC-1α (Affinity Biosciences, Cat. No. AF5395) and GAPDH (Absin, Cat. No. abs132004) were used. The membranes were washed six times, and subsequently incubated with secondary antibodies (CST) (1:2000 dilution in 5% milk/1 × TBST) for 1 h. Proteins were detected using an ECL reagent (Bio-Rad, Shanghai, China) on a Molecular Imager ChemiDoc XRS + System (Bio-Rad). The western blots were quantified using the ImageJ software (National Institutes of Health).