Muscle fiber typing staining kit

Myofibre immunostaining of C2C12 cells was performed using primary antibodies against Myh (catalogue no.: ab172967; Abcam). Staining for muscle fibre-type was performed using the Muscle Fiber Typing Staining Kit (Servicebio), as per manufacturer’s instructions. Images were captured and processed with a Leica DMi8 automated microscope (Leica), and the myofibre area was measured using ImageJ software (RRID: SCR_003070; version 1.51; NIH) (see ESM Methods).

Immunofluorescent staining was conducted for myofibre type in gastrocnemius muscle from miR-193b AAV-treated C57 and sgmiR-193b AAV-treated db/db mice (see ESM Methods). In brief, gastrocnemius muscle and TA slices were subjected to myofibre-type staining using the Muscle Fiber Typing Staining Kit (Servicebio, Wuhan, China), as per manufacturer’s instructions. Rabbit anti-fast myosin skeletal heavy chain antibody (diluted 1:100 in PBS; catalogue no.: GB112130; Servicebio) and mouse anti-slow skeletal myosin heavy chain antibody (diluted 1:100 in PBS; catalogue no.: GB21303; Servicebio) were used as primary antibodies, while Cy3-labelled goat anti-rabbit IgG (catalogue no.: GB21303; Servicebio) and Alexa Fluor 488-labelled goat anti-mouse IgG (catalogue no.: GB25301; Servicebio) (both diluted 1:400) were used as secondary antibodies. DAPI (catalogue no.: P0131-25 ml; Beyotime, Shanghai, China) was used to stain cell nuclei. Images were captured and processed with a Leica DMi8 automated microscope (Leica, Weztlar, Germany) and the myofibre area was measured using ImageJ software (RRID: SCR_003070; version 1.51; NIH, Bethesda, MD, USA) (see ESM Methods for further details).