Immunostaining and Imaging of Mouse Spinal Cords

Spinal cords were recovered and stained as previously described^{60 (link),75 (link)}. Following terminal anesthesia by pentobarbital, mice were perfused transcardially with 10% formalin (Sigma). Spinal cords and brains were removed, post-fixed overnight, transferred to buffered 30% sucrose for 48 h, embedded in O.C.T. Compound (Tissue-Tek, Sakura-Finetek/VWR) and cryostat-sectioned at 30 μm. Serial horizontal sections of spinal cord containing the lesion sites and brain containing the viral injection sites were cut and processed for immunostaining. The following primary antibodies were used: anti-GFAP (DAKO Z0334, 1:1000, free-floating), anti-GAP43 (1:1000, Benowitz lab), anti-Synaptophysin (Synaptic Systems 101004, 1:1000, free-floating), and RFP (1:500, Abcam ab62341, free-floating). BDA tracing was visualized with streptavidin-HRP (1:300, PerkinElmer SAT704A001EA) antibodies plus Cy3-TSA (1:200, PerkinElmer SAT704A001EA). Sections were cover-slipped using Prolong Diamond Antifade Mounting media with DAPI (ThermoFisher) to stain cell nuclei. Each section was imaged on ZEISS LSM 880.