Tissue preparations for immunohistochemistry

Animals were anesthetized with sodium pentobarbital and perfused via the left ventricle with 20–30 ml physiological saline followed by 100 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB: pH 7.4). Brains were immediately removed and immersed in the same fixative for 8 hr. After immersion in 25% sucrose in 0.1 M PB for 3 days at 4°C for cryoprotection, brains were quickly frozen and cut into 20-μm coronal sections using a cryostat (CM3050 S; Leica, Nussloch, Germany). These sections were washed several times with PBS for 5 min each.