Multimodal Neurotracing in Embryos

For DCN neurotracing, embryos were dissected, the spinal cord was exposed via dorsal laminectomy, fixed in 4% paraformaldehyde (PFA) for 2 h, and washed in PBS. For spinal DCN neurotracing, DiI crystals (Molecular Probes) were placed in the dorsolateral region of the spinal cord using glass micropipettes (Renier et al., 2010 (link)). For supraspinal neurotracing, the skull was removed, and DiI crystals were placed into the caudal hindbrain. For sensory DCN neurotracing, DiI crystals were placed in dorsal root ganglia and the ventral roots were cut. Tissue samples were placed in 4% PFA at 37°C for up to 5 weeks for DiI diffusion. Samples were then washed with PBS and embedded in 4–6% agarose for vibratome sectioning (200 μm). Sections were mounted with PBS for confocal microscopy.