Bs 8486r

Co-IP was conducted as previously described. Briefly, the cell lysate was combined with 1.2mL of dilute buffer (lysis buffer), which was incubated with Protein A/G Agarose (sc-2003; Santa, Dallas, TX, USA) and mouse anti-integrin alpha V/beta 5 antibody (sc-81632; Santa, Dallas, TX, USA) overnight at 4 °C. The following day, the agarose beads were collected via centrifugation at 5000 rpm for 15 min at 4 °C. Next, the beads were eluted two to three times and boiled to denature the protein–bead complex. The proteins were separated via SDS-PAGE, electrophoretically transferred onto 0.22 μm nitrocellulose membranes, and immunoblotted with the primary antibodies (rabbit anti-fndc5 antibody (1:1000, bs-8486R; Bioss, Beijing, China), mouse anti-integrin alpha V antibody (1:200, sc-9969; Santa, Dallas, TX, USA), and mouse anti-integrin beta 5 antibody (1:100, sc-398214; Santa, Dallas, TX, USA)).

The muscle tissues were fixed in 4% polyoxymethylene and then embedded in paraffin. H&E staining procedures were performed as described previously (Feldman and Wolfe, 2014 (link)). For immunofluorescence (IF) and immunohistochemistry (IHC) staining, 5-µm-thick paraffin-embedded muscle sections were processed using a standard histological protocol. The paraffin slides were deparaffinized, rehydrated through graded alcohols to deionized water, and subjected to antigen retrieval in a pressure cooker, at 100°C for 20 min. Blocking was performed using Dako protein Block, 3% BSA (Merck Millipore, Massachusetts, Germany) in TBST, and 5% goat serum in TBST. The primary antibodies, including anti-FNDC5 antibody (bs-8486R, Bioss, Beijing, China) (1:1,000), anti-GDF8/myostatin antibody (ab203076, Abcam, Cambridge, United Kingdom) (1:1,000), antimyosin heavy chain (MyHC)-IID antibody (bs-5885R, Bioss, Beijing, China) (1:1,000), and anti-MYH7 (MyHC (slow)) antibody (GB111857, Servicebio, Wuhan, China) (1:1,000), were incubated with the tissue slides overnight at 4°C. Slides were washed in TBST and incubated with secondary antibodies (ab150115, ab150077, and ab6721, Abcam, Cambridge, United Kingdom) diluted in 5% skimmed milk in TBST for 1 h at room temperature. Confocal microscopy was applied to evaluate the expression of FNDC5 and Mstn.