Immunofluorescence staining and imaging

Freshly isolated TA muscles were fixed in 4% PFA at 4 °C for 60 min. After fixation, tissues were washed in PBS three times, dehydrated in 30% sucrose overnight at 4 °C, embedded in OCT and flash frozen with liquid nitrogen. 10 µm sections were collected on glass slides and stored at −20 °C. For immunofluorescence staining, slides were put in a fume hood to air dry, followed by a brief PBS wash to remove OCT. Slides were blocked and permeabilized with 1% BSA and 0.1% Triton X-100 in PBS for 30 min, followed by incubation with primary antibodies overnight at 4 °C in the dark. Primary antibodies used in this study are listed in Supplementary Table ². On the following day, the slides were washed in PBS for 10 min, 3 times to remove unbound primary antibodies, and then incubated with fluorescence-conjugated secondary antibodies for 60 min at room temperature in the dark. After washing in PBS for 5 min, 3 times to remove secondary antibodies, the sections were stained with DAPI (4′,6-diamidino-2-phenylindole) and mounted with Aqua-Poly/mount (Polysciences). For detection of Pax7 with anti-Pax7 (DSHB), a tyramide staining strategy was used. Cryosections were stained using TSA Kit per manufacturer’s instructions (Akoya). For each experiment, the TA muscles were derived from at least 5 mice. Over 30 serial cryosections were collected, and the slices with maximum cross-sectional area were selected for immunofluorescence staining and quantification. For visualizing myofibers or MuSCs, myofibers or MuSCs were fixed using 4% PFA, blocked and permeabilized using 1% BSA and 0.1% Triton X-100 in PBS, and stained with primary antibodies against tdT, Pax7 or MyHC, then washed and stained with fluorescence-conjugated secondary antibodies. Counterstain of nuclei with DAPI was performed at the end of the staining (Invitrogen). Images were acquired on Olympus fluorescence microscope (BX53) or Leica TCS SP8 confocal microscope. ImageJ software was used to analyze the collected images.