Cresyl violet nissl stain

For Ki67 staining, an avidin-biotin complex (Elite ABC kit, Vector, Burlingame, CA, USA) method was employed with a primary polyclonal rabbit anti-Ki67 antibody (1:200; NCL-Ki67p, Novacastra, Newcastle upon Tyne, UK), a secondary biotinylated goat anti-rabbit IgG (1:300, Jackson Immunoresearch Laboratories Inc., West Grove, PA, USA) and 3, 3^′-diaminobenzidine (DAB) as the chromogen. Sections were counterstained with cresyl violet nissl stain (Sigma, Germany). For DCX immunostaining, an ABC protocol similar to that employed by Rao and Shetty (2004 (link)) was used. A primary goat polyclonal anti-DCX antibody (1:200, sc-8066, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used with a biotinylated rabbit anti-goat IgG (1:300; Jackson Immunoresearch Laboratories Inc.) and DAB as the chromogen.

Post-injury lesion volume was quantified as previously described^{15 (link)}. Briefly, coronal brain sections were stained using Cresyl violet (Nissl stain; Sigma-Aldrich). The area of lesioned structures was expressed as a percentage of the volume of corresponding structures in the contralesional hemisphere. Lesion volume was measured using ImageJ (National Institutes of Health, Bethesda, MD, USA); the percentage of the lesion volume was calculated as (contralesional cortex–ipsilesional cortex)/contralesional cortex. To assess CST destruction volume, transverse cervical cord sections were stained using anti-PKCγ antibody. CST area was expressed as a percentage of the PKCγ-immunoreactive area of the uninjured CST (C4–C7). PKCγ staining intensity was measured using ImageJ; the percentage of the lesion volume was calculated as (uninjured CST–injured CST)/uninjured CST.