Western Blotting Analysis of Muscle Markers

The mice were perfused with 0.1 M PBS, and their tissues were rapidly dissected. The TA tissues were snap-frozen using cooled isopentane and stored at −80 °C until needed. To prepare the samples for analysis, equal amounts of total protein homogenates were loaded onto a polyacrylamide gel and electroblotted onto a PVDF membrane (Millipore, Burlington, MA, USA), following the method previously described in [25 (link)]. The membranes were then immunoblotted with primary antibodies, followed by HRP-conjugated secondary antibodies from Santa Cruz Biotechnology (Dallas, TX, USA). The Luminata Forte Western Chemiluminescent HRP Substrate from Millipore (Burlington, MA, USA) was used to develop the blots, and the Chemi-Doc XRS system from Bio-Rad (Hercules, CA, USA) was used for visualization. The immunoreactivity was normalized to the total amount of protein loaded, as determined by Ponceau staining. The primary antibodies used in the experiment were mouse anti-Pax7 (1:1000; DSHB, Iowa City, IA, USA), rabbit anti-MyoD (1:1000; Proteintech, Rosemont, IL, USA), mouse anti-MyoG (1:130; DSHB, Iowa City, IA, USA), and CD206 (1:500; Abcam, Cambridge, UK).