Eclipse te2000 s system

The satellite cells were collected when they converged to approximately 60% or induced differentiation for 24 or 48 hr, washed twice with phosphate‐buffered saline (PBS), and fixed in 4% paraformaldehyde for 15 min. Then, the satellite cells were washed twice with PBS, incubated in an ice‐cold 0.25% Triton X‐100 at room temperature for 10 min, and again washed thrice. The cells were incubated in blocking solution (3% bovine serum albumin, 0.3% Triton X‐100, 10% fetal bovine serum in PBS) at room temperature for 2 hr and then incubated in primary antibody at 4°C overnight. The primary antibodies for immunofluorescence staining are shown in Supporting Information Table S1. The cells were washed thrice with PBS and then incubated with anti‐mouse IgG (H+L), F (ab’) 2 Fragment (Alexa Fluor^® 555 Conjugate; CST, USA, 4409), and anti‐rabbit IgG (H+L), F(ab’) 2 Fragment (Alexa Fluor^® 488 Conjugate; CST, 4412) for 2 hr. The cell nucleus was washed thrice with PBS and stained with 4′, 6‐diamidino‐2‐phenylindole (DAPI) (Wei et al., 2013). Images were captured using a Nikon Eclipse TE2000‐S system (Nikon, Japan).

The gastrocnemius muscle was dissected form pirfenidone‐treated mice and mice of different ages (postnatal Day 1, Day 8, Week 2, Week 4, Week 6, Week 8, Week 10, Week 12, Week 24, and Week 52). After fixation with 4% paraformaldehyde, the skeletal muscle samples were embedded in paraffin, and 4‐um‐thick serial sections were obtained. After deparaffinization, citric acid buffer (PH6.0) microwave antigen retrieval method was used. Additional immunofluorescence staining was performed according to the cell immunofluorescence experiment. Images were captured using both Nikon ECLIPSE TE2000‐S system (Nikon) and confocal microscopy (ZEISS, Germany).