Neuromuscular Junction and CNS Analysis in SOD1^G93A Mice

SOD1^G93A mice and wild-type littermates were sacrificed by inhalation of CO₂ for muscle analysis. The extraocular muscles and lumbrical muscles (from the plantar surface of the hind-paw) were dissected in 0.1 M PBS and fixed in 4% PFA (Sigma-Aldrich) for 10 min for NMJ analysis or snap frozen in 2-Methylbutane (Sigma-Aldrich) on dry ice for immunoblotting. Only muscles innervated by CNIII were included in the extraocular analysis (superior rectus, inferior rectus, medial rectus, and inferior oblique). Staining for NMJ analysis was done as previously described6 (link) using antibodies detailed in Table 1. For CNS immunohistochemistry, animals were anesthetized with avertin (2,2,2-Tribromoethanol; Sigma-Aldrich) and perfused intracardially with PBS followed by 4% PFA. Brains and spinal cords were dissected and postfixed (for 3 hours and 1 hour, respectively), cryoprotected in sucrose and sectioned (30 μm). All CNS tissues were stained as previously described12 (link) using the following primary antibodies (Table 1) and counter stained with NeuroTrace 435/455 Blue Fluorescent Nissl Stain (1:200 in PBS; Life Technologies) for 30 min. Tissues were imaged on a Zeiss LSM700 confocal microscope.