Brain Tissue Preparation and Immunohistochemistry

Isolated fresh brains were sliced into 4-mm thick tissue sections using the coronal plane as a reference and fixed in 4% paraformaldehyde for 72 h. Then, the fixed brain tissues were sequentially dehydrated in 15–30% sucrose solution, sliced further into 40-µm thick sections using a SM2000R microtome (Leica, Germany), and stored in ethylene glycol at -20 °C.
Subsequently, the sections were subjected to immunohistochemical staining. The primary and secondary antibodies used in this study were listed in Supplementary Table 2. Briefly, hydrogen peroxide was used to quench the activity of endogenous peroxidase. Citrate buffer was used for antigen repair. The sections were incubated with primary antibodies at 4°C for 16 h and then with horseradish peroxidase (HRP) -labeled secondary antibodies. The avidin-biotin-peroxidase complex (prepared from VECTASTAIN ABC Reagent kit) and substrate chromogen 3,3’-diaminobenzidine tetrahydrochloride hydrate (DAB) were used to visualize the immune complex. The whole brain images were captured under a VS200 Virtual Slide Microscope (Olympus, Japan).