Immunohistochemistry for myelin basic protein (MBP)

To obtain accurate data for immunohistochemistry, free-floating sections from all rats were processed carefully under the same conditions as described previously [30]. For each animal, tissue sections were selected with reference to a rat brain atlas [31], between 3.00 and 4.08 mm posterior to the bregma. Ten sections, 90 µm apart, were sequentially treated with 0.3% hydrogen peroxide (H₂O₂) in PBS for 30 min and 10% normal horse serum in 0.05 M PBS for 30 min thereafter. The sections were then incubated with a rabbit anti-MBP antibody (1:1,000, Abcam, Cambridge, UK) overnight at 25℃, before being sequentially treated with either biotinylated goat anti-rabbit IgG, or a streptavidin-peroxidase complex (1:200, Vector, Burlingame, CA). Sections were visualized by the reaction with 3,3-diaminobenzidine tetrachloride (Sigma) in 0.1 M Tris-HCl buffer (pH 7.2). The sections were then dehydrated, before being mounted onto gelatin-coated slides, using Canada balsam (Kanto, Tokyo, Japan).

In order to establish the specificity of primary antibody, procedure included the omission of the anti-MBP antibody, goat anti-rabbit, and substitution of normal goat serum for the anti-MBP antibody. As a result, immunoreactivity disappeared completely in tissues. All experiment procedures in the present study were performed under the same circumstance and in parallel.