Comprehensive Brain Blood Vessel Imaging

DyLight594-lycopersicon esculentum lectin (1 mg/ml, 0.2 ml per mouse; Vector Labs, United States) was administered via the tail vein 15 days after MCAO. The dye was allowed to circulate for 10 min. Mice were anesthetized and then transcardially perfused with normal saline and 4% PFA in turn. The brains were removed and placed in 4% PFA for 24 h. After embedding in resin (Gong et al., 2016 (link); Gang et al., 2017 (link)), the whole brain specimens were imaged continuously using the fMOST system (BioMapping 5000; OEBio Inc., Wuhan, China). All the image tiles were obtained with a time-delay integration line scan charge-coupled device (Zhou YF. et al., 2019 (link)), and the images had a voxel size of 0.35 μm × 0.35 μm × 1 μm. Afterwards, the original images were preprocessed (Gong et al., 2016 (link)), and visualization and 3D data block contour rendering were performed using Amira software (version 5.2.2; FEI, France). The blood vessels of each data block (size of 400 μm × 400 μm × 400 μm) were tracked semi-automatically, and the VLD and FVV were calculated.