Neuroanatomical Tracing of Corticospinal Projections

The animals were deeply anesthetized and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer. Then, the brain and cervical spinal cord were dissected out and post-fixed in the same fixative solution overnight at 4°C. The samples were cryoprotected in 30% sucrose in PBS and then embedded in an optimal cutting temperature (OCT) compound (Sakura Finetek). The brain and cervical spinal cord were cut serially into 50- and 20-μm thick coronal sections, respectively. To plot the lesion area in the sensorimotor cortex, coronal brain sections were stained with Nissl staining (0.1% cresyl violet). For BDA staining, sections of the cervical spinal cord were incubated in 0.3% Triton X-100 in PBS for 4 h, followed by Alexa Fluor 568-conjugated streptavidin (1:400, Invitrogen) in 0.1% Tween 20 in PBS for 2 h at room temperature as described previously (Ueno et al., 2012 (link), 2018 (link)).