Luxol Fast Blue (LFB) Staining

After anesthetization with 3% pentobarbital sodium (0.2 ml/kg), rats were transcardially perfused with 0.9% saline followed by 4% paraformaldehyde (Solarbio; cat. no. P1110). The brain was removed and immersed in 4% paraformaldehyde for 24 h. The corpus callosum was embedded in paraffin and cut into 5-μm–thick coronal sections that were deparaffinized in xylene, absolute ethanol, and 75% ethanol before incubation in LFB solution (Servicebio; cat. no. G1030) for 4 h at 60°C. The sections were placed in 0.05% lithium carbonate solution followed by 70% ethanol to reduce background staining, then mounted on slides and sealed with neutral balsam for light microscopy examination (Eclipse E100, Nikon, Tokyo, Japan). The degree of demyelination was quantified with Image-Pro Plus v.6.0 software (Media Cybernetics, Bethesda, MD, USA).