TA muscles were harvested and fixed with 4% paraformaldehyde (PFA) for 1 h at room temperature and then immersed in 30% sucrose overnight at 4 °C, as described in our previous study [14 (link)]. On day 2, the TA muscles were cryopreserved in an optical cutting temperature (OCT) compound (Tissue Tek) at −80 °C. TA muscle samples were sliced into 5-μm-thick frozen cross-sections using a Leica CM3050 cryostat. The muscle sections were incubated with primary antibodies including human-specific dystrophin (NBP2-79783, 1:200, Novus Biologicals, Centennial, CO, USA), human nuclear antigen (NBP2–34342, 1:100, Novus Biologicals, Centennial, CO, USA), CX3CL1(1:100, ABclonal, Woburn, MA, USA), VCAM-1(1:100, ABclonal, Woburn, MA, USA) and Myh3 (DSHP, 1:100) at 4 °C overnight, respectively, and anti-rabbit/mouse/rat secondary antibodies conjugated to Alexa Fluor 594 (Life Technologies) at room temperature for 1 h. Images were taken using a confocal microscope (FV1000, Olympus, Tokyo, Japan). For cell-engraftment quantification, four sections at 150 μm intervals in each TA muscle were analyzed. The number of muscle fibers in a cross-section area was measured using ImageJ with the colocalization plugin (NIH).
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