Muscle Regeneration Immunofluorescence Protocol

For immunofluorescence staining of Pax7, EdU, Myog and eMyhc, the Gas muscles were harvested at day 3. Immunofluorescence staining on frozen muscle sections was performed in accordance with previous reports^{21 (link)}, and images were visualized using a confocal laser scanning microscope (Zeiss, LSM800, Germany). The following dilutions were used for each antibody: Pax7 (Developmental Studies Hybridoma Bank; USA; 1:20), eMyhc (Developmental Studies Hybridoma Bank, USA; BF-G6; 1:100), Myog (Santa Cruz Biotechnology, USA; sc-12732; 1:20). To detect the EdU incorporation, the sections were performed using the Life Technologies Click-iT Kit according to the manufacturer’s instructions, and images were photographed using a confocal laser scanning microscope (LSM800; Zeiss). For H&E staining, the Gas muscles were harvested at day 0, 3, 7, and 15 after CTX injection. H&E of muscle sections was performed according to previous reported methods^{21 (link),45 (link)}, and the cross-section area of individual myofibers was visualized using Olympus DP80 upright Metallurgical Microscope (Olympus Corporation, Japan) and qualified using ImageJ software.