Immunohistochemistry of Brain Microvasculature

For immunohistochemistry (n = 4 for each group), coronal fresh frozen sections were sliced starting from the optic chiasma in 10 μm thick using a cryostat (CM1800, Leica, Bensheim, Germany). The slicing method was the same as above. The sections were incubated with mouse anti-CD31 (1:50, Thermo Fisher Scientific, MA1-80069, Waltham, MA, United States) diluted in PBS overnight at 4°C after blocking with bovine serum albumin. Then the samples were incubated with a biotinylated secondary antibody followed by avidin–biotin–peroxidase complex. Positive staining was visualized with diaminobenzidine. The images were captured by a digital camera connected to a microscope (BX512DP70, Olympus, Tokyo, Japan). Five fields were randomly selected for each rat. The number of open microvessels and microvessels with perivascular edema was analyzed with Image-Pro plus 5.0 software (IPP, Media Cybernetic, Bethesda, MD, United States). Five fields of the hippocampus and cortex region were randomly selected and examined separately for each animal (Gu et al., 2018 (link)). The microvessels that had the CD31-positive endothelium sticking together without any lumen were defined as closed microvessels. The percentage of microvessels with perivascular edema and closed microvessels in each field was calculated.