Ab51263

Cells cultured in 12-well plates were fixed in 4% paraformaldehyde for 10 min, followed by permeabilization in 0.5% Triton X-100 for 15–20 min. The cells were blocked with 4% BSA in Tris-buffered saline with Tween (TBST) for 1 h. Then, the cells were incubated with primary antibodies overnight at 4°C. Afterwards, the cells were washed in PBS three times and incubated with secondary antibodies for 1 h at room temperature. Finally, the cells were washed three times in PBS, and the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1:1000 in PBS). The antibodies used were as follows: anti-MyoD antibody (Abcam, ab212662), anti-Pax7 antibody (Thermo Fisher Scientific, PA1117), anti-Fast Myosin Skeletal Heavy chain antibody (Abcam, ab51263), anti-mouse IgG (H+L), F (ab')2 fragment (Alexa Fluor 555 conjugate) (Cell Signaling, #4409), and anti-rabbit IgG(H+L), F (ab')2 fragment (Alexa Fluor 488 conjugate) (Cell Signaling, #4412). Immunostaining images were obtained via fluorescence microscopy on an inverted microscope (Nikon, Tokyo, Japan).

Immunofluorescent staining for fiber-specific SC content (PAX7⁺ cells) and activation status (quiescent PAX7⁺/MYOD⁻, activated PAX7⁺/MYOD⁺, and differentiating PAX7⁻/MYOD⁺) are described previously (8 (link), 33 (link)–36 (link)) and expressed per 100 fibers. All staining procedures were verified for specificity using negative controls for primary (primary only) and secondary (secondary only) antibodies. For quantification, PAX7 (anti-PAX7 Mouse, DHSB, neat; Alexa Fluor 594 goat anti-mouse, 1:500) and/or MYOD (anti-MYOD 5.8 A Mouse, DAKO, 1:100; goat anti-mouse biotin, 1:200, and streptavidin 488, 1:200) was overlayed with DAPI (Sigma-Aldrich, 1:20,000) and examined with laminin (anti-Laminin Rabbit, Abcam ab11575, 1:500; Alexa Fluor 647 goat anti-rabbit 1:500) or Wheat Germ Agglutinin (WGA; Wheat Germ Agglutinin, Invitrogen W32466, 1:200) to determine the appropriate SC location, myosin heavy chain I (anti-MHCI Mouse, DHSB A4.951, neat; Alexa Fluor 488 goat anti-mouse, 1:500) and II (anti-MHCII Rabbit, Abcam ab51263, 1:1,000; Alexa Fluor 647 goat anti-rabbit, 1:500) to determine fiber type-specific associations and expressed per 100 fibers. Images were taken on a Nikon Eclipse Ti Microscope (Nikon Instruments) with a high-resolution Photometrics CoolSNAP HQ2 fluorescent camera (Nikon Instruments, Melville, NY) at a ×20 objective. Analyses were performed in a blinded fashion.

Gastrocnemius muscles were collected and fixed with formalin solution neutral buffered 10%. After dehydration and paraffin embedding, tissue sections were cut (5 μm-thick). Slides, dewaxed and hydrated, were stained with hamatoxylin and eosin (H-E) or reacted with mouse monoclonal anti-fast myosin skeletal heavy chain (1:1000; ab51263, Abcam, Cambridge, UK) and anti-slow myosin skeletal heavy chain (1:250; ab11083). Slides were analyzed under a light microscope (Eclipse E600, Nikon, Tokyo, Japan) and images captured (magnification 20X) by a digital camera (DXM1200F, Nikon) connected to the ACT-1 software (Nikon). Then, the cross-sectional area (CSA) was measured using the ImageJ software 2018 (NIH, Bethesda, Maryland, USA), as reported [21 (link)].

For IF staining, the wax blocks were cut into 4 μm on the HistoCore BIOCUT (Leica Biosystems, Shanghai, China) and baked in an oven at 60 °C for 3 h. Antigen retrieval was performed using a sodium citrate antigen retrieval solution (Solarbio, Beijing, China, C1032). Muscle sections were blocked with 3% BSA for 40 min at room temperature and incubated with primary antibodies specific for MyHC-I (Proteintech, Wuhan, China, 22280-1-AP), Fast Myosin Skeletal Heavy Chain (Abcam; ab51263), and Laminin (Abcam, ab11575) overnight at 4 °C, followed by IgG H&L (FITC) (Abcam, ab6717), IgG H&L (Cy3) (Abcam, ab97035) secondary antibodies for 1 h at room temperature. Fluorescence images were collected by a confocal laser microscope (Zeiss, Jena, Germany, LSM880) and processed by Image J software or Zen 2.6 lite.

The differentiated C2C12 myoblasts were washed with PBS, fixed with 4% paraformaldehyde for 20 minutes, and permeabilized with 0.2% Triton X-100 for 10 minutes at room temperature (22°C ± 3°C). Then, nonspecific antibody binding was blocked with 5% goat serum (Bioss, Beijing, China) for 2 h and subsequently incubated with anti-MyHC (ab51263, Abcam, USA) overnight at 4°C. The next day, the C2C12 myoblasts were incubated with Alexa Fluor® 488 secondary antibody (ab150113; Abcam) for 1h. Finally, the cells were observed on a fluorescence microscope (Olympus, Kyoto, Japan) [22 (link)].