Bf 45

For sterile muscle injury, mice were anesthetized with isoflurane and 50 μL cardiotoxin (12 × 10^–6 M, 217503, Millipore) injected in the TA muscle. Muscles 8 days after injury were snap frozen in nitrogen-chilled isopentane. 8 μm cryosections were cut and stained with H&E. For each analysis, more than 10 slides (per condition/group) containing 6 muscle sections/sample were used, and myofibers in the injured area were counted and measured with a Mirax digital scanner and HALO software (68 (link)). For adult mouse studies, muscles were fixed in 10% formalin and embedded in paraffin for sectioning. Laminin staining (RB-082, Thermo Fisher Scientific) was used to outline fibers, and picrosirius red was used for collagen accumulation. Image analysis was performed using ImarisX64 software (Bitplane AG), and fiber sizing was calculated using minimal Feret’s diameter (69 (link)). For after weaning juvenile mouse studies, cryosections from soleus muscles were mounted in ProLong antifade mountant with DAPI (Thermo Fisher Scientific). Fiber type, size, and central nucleation were quantified with analysis via SMASH (70 (link)) and ImageJ (NIH). Antibodies used were mouse anti-MHC2A (1:50, SC-71, Developmental Studies Hybridoma Bank) and mouse anti-eMHC (1:10, BF-45, Developmental Studies Hybridoma Bank).

The mouse monoclonal antibodies specific for eMyHC (BF45) and type 1 MyHC (BAF8) were purchased from Developmental Studies Hybridoma Bank. Tissues were cryo-sectioned at 10 μm and immunostained using antibodies specific for eMyHC (1:10) or type 1 MyHC (1:5) overnight at 4 °C and processed as before^{69 (link)}. For fiber size measurements, the tissue sections were stained with WGA, (fluorophore conjugated, 1:100, Cat.# W11262, Life Technologies). Images were obtained using a Zeiss LSM 510 on Zeiss Axiovert 100 M Base and processed using NIS Elements. The minimal “Feret’s” diameter variance coefficients of the muscle fiber size was calculated on the WGA stained sections using the ImageJ 1.43u program.