Brain Tissue Preparation

Four days after the injections, the animals were anesthetized, transcardially perfused with 0.05 M PBS, and then fixed with ice-cooled 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The brain tissue was extracted, postfixed in 0.1 M PB containing 4% paraformaldehyde for 20 h at 4 °C, and then saturated with a 30% sucrose in 0.05 M PBS solution for 3 days at 4 °C for cryoprotection. The samples were embedded with optimal cutting temperature (OCT) compound and cut into serial 30-μm-thick coronal sections with a cryostat (Leica Biosystems, Wetzlar, Germany). The tissue sections were stored in a cryoprotectant (25% ethylene glycol, 25% glycerol, and 0.05 M PB) at 4 °C until they were needed for histology.