Myelin basic protein (MBP) labeling in the hippocampus and corpus callosum

Mice were anesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg) ip and perfused transcardially with 10 mL PBS (pH 7.4) followed by 40 mL 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7). Brains were removed and cryoprotected in 30% sucrose in PBS. The brains were frozen in powdered dry ice, and coronal, 25-μm-thick sections were prepared with a freezing microtome (Leica). The sections were collected in freezing solution (30% ethylene glycol; 25% glycerol; 0.05 M phosphate buffer) and stored at −20°C until used. The sections were washed three times in PBS and then in 0.5% Triton X-100 + 0.5% H₂O₂ in PBS 15 minutes and rinsed again in PBS three times. The nonspecific antibody binding was blocked by treatment in 2% normal horse serum in PBS for 20 minutes followed by incubation in rabbit anti-MBP primary antibody (1:250, AB980; Merck-Millipore) for 24 hours and washed in PBS. Fluorescein -conjugated antirabbit IgG (1:250; Jackson ImmunoResearch) was applied for 1.5 hours and the sections were washed again in PBS three times. The sections were mounted on glass slides and coverslipped with Vectashield mounting medium (Vector). The ImageJ software (National Institutes of Health) was used to measure total density of the whole hippocampus (n = 6).