Polink 2 plus polymer hrp detection kit

For immunohistochemical studies, mice were anesthetized and then perfused through the aorta with 0.9% normal saline followed by phosphate buffer containing 4% paraformaldehyde. Brains were removed and postfixed in perfusate overnight and then immersed into 30% (wt/vol) sucrose liquid diluted with PB twice for about 3 days. Once sinking to the bottom of the tubes, brains were snap frozen at −80 °C and then cut for slices (30 μm) with a freezing microtome. The brain slices were washed with PBS containing 0.1% Triton X-100 (PBST) for 3×5 min, followed by 3% H₂O₂ diluted in PBS for 30 min. After washing with PBST for 3×5 min, the brain slices were blocked with 5% BSA for 30 min at room temperature. Then slices were incubated with primary antibodies overnight at 4°C. After washing with PBST three times, immunoreaction was developed using the Polink-2 plus® Polymer HRP Detection kit (ZSGB-BIO, PV-9001/PV9002) and visualized with diaminobenzidine (brown color). Slices were sequentially dehydrated in 50%, 75%, 95% and 100% ethanol for 6 times (20 min each) and cleared in xylene for 3 times (30 min each) and cover-slipped with Permount solution. Images were obtained with a microscope (Olympus BX60, Tokyo, Japan).