Bs 2539r

To block endogenous peroxidase activity, 5 μm-thick deparaffinized sections were incubated with 1% H₂O₂ for 30 min at room temperature. To observe the morphological characteristics and detect protein expression in the quadriceps muscle, the quadriceps muscle tissues were reacted overnight at 4 °C with anti-TWEAK (ab37170; 1:400; Abcam, Cambridge, UK), anti-NF-κB (sc8008; 1:300; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-PGC-1α (bs-1832R, 1:300, BIOSS, Beijing, China) and anti-MuRF1 (bs-2539R; 1:300; BIOSS, Beijing, China) antibodies. Samples were then washed with PBS and incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (PV-8000; 1:200; ZSGB-BIO, Beijing, China) for 1 h at room temperature. Following the removal of non-reacted secondary antibodies by washing with PBS, samples were incubated with 3,39-diaminobenzidine (DAB; Sigma-Aldrich; Merck KGaA) in a DAB-4HCl-H₂O₂ solution to visualize immunolabeling. Some sections were also counterstained with hematoxylin and eosin and mounted with a coverslip with neutral resins. Immunohistochemical analysis was performed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). A total of 4–5 images that were positive for protein expression were randomly selected and their integral luminosity values and average optical density were assessed using a light microscope.

The differentiated C2C12 myotubes were stimulated with CSE (3%, 1.5%, and 0.75%) for 24 and 48 h. Before harvest, cells were treated with Monensin (BioLegend, California, United States) for another 4–6 h. Then, cells were incubated with fluorescence-conjugated antibodies, including rabbit antimouse myostatin antibody (ab203076, Abcam, Cambridge, United Kingdom), rabbit antimouse FNDC5 antibody (bs-8486R, Bioss, Beijing, China), antimouse PGC-1α antibody (AF7736, Beyotime, Shanghai, China), and antimouse MuRF1 antibody (bs-2539R, Bioss, Beijing, China). The second antibodies included PE antirabbit IgG (12-4739-81, eBioscience, United States) and isotype PE antirabbit IgG (400707, BioLegend, United States). The cells were then fixed in 0.5 ml/tube Fixation Buffer (BioLegend) in the dark for 20 min at room temperature. Permeabilization was performed using Intracellular Staining Perm Wash Buffer (BioLegend). Flow cytometry data were acquired on a CytoFlex LX flow cytometer (Beckman Coulter, CA, United States) and analyzed using FlowJo (Tree Star, San Carlos, California, United States).