Fluorescence camera microscopy

Brain samples were fixed with 4% paraformaldehyde overnight at 4°C, and sliced into 3-μm-thick sections. The sections were permeabilized with 0.1% Triton X-100 for 10 min, and then washed with PBS twice. Sections were incubated in a dark box with VEGFA primary antibodies (1:150) for 24 h at 4°C. After that, the sections were again washed with PBS twice, and incubated with appropriate fluorescence–conjugated secondary antibodies for 2 h at room temperature. DAPI reagent was used to stain the cell nucleus. All of the images were obtained using fluorescence camera microscopy (Leica, Wetzlar, Germany), and positive expression was quantified using the Image-Pro Plus software [20 (link)].

As with immunohistochemistry staining, sections of HUVECs were incubated in a dark box with HIF primary antibody (ab197493, Abcam, Danvers, MA), and appropriate fluorescence-conjugated secondary antibody (Beijing Zhongshan Jinqiao Biotechnology Co. Ltd., Beijing, China). DAPI reagent was used to stain the cell nucleus. All of the images were obtained using fluorescence camera microscopy (Leica, Wetzlar, Germany). The results were based on the five areas of positivity observed, and the proportion of positive staining area was calculated by Image J IHC Profiler software.