Quantifying Activated MuSCs in Transplanted Muscle

We collected the culture progeny of MuSCs from aged Myf5^nLacZ/+/Luciferase double-transgenic mice^{23 (link),43 (link)} by incubation with 0.1% trypsin in PBS for 2 min at 37 °C and transplanted them into tibialis anterior muscles of hindlimb-irradiated NOD/SCID mice. One month after transplant, we injected notexin to damage recipient muscles and activate MuSCs in vivo. Four days later, we collected, fixed, and cryosectioned recipient muscles, as described above. We performed immunohistological analysis of transverse tissue sections to detect β-galactosidase⁺ cells (indicating a donor-derived cell expressing Myf5, a marker of MuSC activation) in the satellite cell position within the myofiber basal lamina, as defined by laminin staining. We stained sections with anti-Laminin (Millipore, clone A5, catalog # 05-206, 1:250) and anti-β-galactosidase (Invitrogen, catalog # A11132, 1:100) primary antibodies and then with appropriate secondary antibodies (Invitrogen). We counter-stained nuclei with Hoechst 33342 (Invitrogen). We acquired images with an AxioPlan2 epi-fluorescent microscope (Carl Zeiss) with Plan NeoFluar 10×/0.30NA or 20×/0.75NA objectives (Carl Zeiss) and an ORCA-ER digital camera (Hamamatsu). We captured digital images in OpenLab software (Improvision) and assembled them using Photoshop software (Adobe) with consistent contrast adjustments across all images.