Sp immunohistochemistry kit

Mice were intraperitoneally anesthetized with 1.5% pentobarbital sodium after 24 hours of reperfusion. The heart was exposed and transcardially perfused with cold 0.9% saline solution, followed by 4% paraformaldehyde. The brain was removed, fixed in 4% paraformaldehyde overnight, cut into 5-mm-thick coronal sections at the 2–5-mm area behind the anterior fontanelle, and then coated with poly-lysine plates. After routine dewaxing, hydration, and microwave antigen retrieval of sections, ionized calcium-binding adaptor molecule 1 (Iba1) (rabbit monoclonal antibody; ab178846, 1:500; Abcam, Cambridge, UK) was immunohistochemically stained by diaminobenzidine using an SP immunohistochemistry Kit (rabbit; Beijing Zhongshan Golden Bridge Biotechnology, Beijing, China). Instead of primary antibody, phosphate-buffered saline solution was used as the negative control. An Eclipse Ci-L microscope (Nikon, Tokyo, Japan) was used for observation. Five high magnification fields (400×) in the hippocampus on the ischemic side were randomly selected in each mouse. Image pro plus 5.0 software (Media Cybernetics) was used to count Iba1-positive cells.