Immunohistochemical Analysis of Angiogenesis Markers

Slices were dewaxed in xylene twice, and then rehydrated in gradient ethanol solution. After antigen recovery using the microwave method in citric acid solution, the sections were blocked in 5% BSA in TBST for 1 h at room temperature, then incubated with the primary antibodies against CD31 (1:250, A19014), VEGFA (1:200), PDGFRB (1:200), VWF (1:200), PDGFB (1:200, A1195), and VEGF (1:200, A16703) purchased from ABclonal (Wuhan, China) and Ki67 (ready-to-use, MAB-0672, Maixinbio, China) at 4°C overnight. The secondary antibody in the streptavidin-biotin kit (#KIT-9720; Maixin-Bio, China) was added to the slides and incubated for 1 h. DAB substrate solution was used to show the positive stain, and the sections were counterstained with hematoxylin. The sections were photographed, and two individuals (G. Y. and Y. C.) independently evaluated all these slides and analyzed the final results using a semiquantitative scoring system as previously described (Gao et al., 2021 (link)).