Muscle Cryosectioning and Immunostaining

Muscles were harvested and used for cryosectioning and immunofluorescence staining as described earlier⁴⁴ . TA muscles transplanted with GFP positive MuSCs were fixed in 2% PFA for 3 h at room temperature (RT) and a sucrose gradient was applied for three consecutive nights (10% → 20% → 30% sucrose in ddH₂O) before freezing in liquid nitrogen. Sections were not additionally fixed for eMHC staining. Cross sections from transplanted muscles were additionally fixed in 2% PFA for 30 min before immunofluorescence staining. Afterwards, antigen retrieval was performed in a decloaking chamber (Biocare Medical) for 10 min at 95 °C using 1 x HIER antigen retrieval buffer (Abcam) before the staining was proceeded. Primary and secondary antibodies are listed in Table 1 and 2 in the supplemental information. Microscopic images were acquired on an upright Axio imager microscope from Zeiss with a 20x objective.
Masson Trichrome staining was performed with the HT15-1KT kit (Sigma Aldrich) according to the manufacturer’s protocol and sections were analyzed using Qupath 0.2.3^{45 (link)}.