Sections were immersed in 3% hydrogen peroxide for 15 min. Nonspecific antigen blocking was performed in 2% bovine serum albumin (BSA) for 1 hour. Sections were then incubated overnight at 4 °C with rabbit anti-Ki67 (1:200, Abcam), rabbit anti-CD31 (1:200, Bioworld), or rabbit anti-HIF-1a (1:100, Boster), then incubated with biotinylated anti-rabbit IgG (1:100, Santa Cruz) for 1 hour at 37 °C, with the avidin-biotinperoxidase complex (1:100, Vector Laboratories) for 1 hour at 37 °C. Immunoreactivity was visualized with diaminobenzidine staining and imaged under a microscope.
To explore the promoting vessel normalization effects of ORI, we performed immunofluorescence double labeling. Sections were incubated in rabbit anti-CD31 (1:200, Bioworld), with mouse anti-a-SMA (1:100, Boster) for 1 hour at 37 °C. For macrophage polarization assessment, sections were incubated in mouse anti-CD206 (1:100, Santa Cruz) for 1 hour at 37 °C. Antibodies were then used: the fluorescein 488-conjugated donkey anti-rabbit antibody (1:1000, Jackson Immunoresearch, USA), or Cy3-conjugated sheep anti-mouse antibody (1:1000, Jackson Immunoresearch). The visual data were scanned using a fluorescence microscope.
To explore the promoting vessel normalization effects of ORI, we performed immunofluorescence double labeling. Sections were incubated in rabbit anti-CD31 (1:200, Bioworld), with mouse anti-a-SMA (1:100, Boster) for 1 hour at 37 °C. For macrophage polarization assessment, sections were incubated in mouse anti-CD206 (1:100, Santa Cruz) for 1 hour at 37 °C. Antibodies were then used: the fluorescein 488-conjugated donkey anti-rabbit antibody (1:1000, Jackson Immunoresearch, USA), or Cy3-conjugated sheep anti-mouse antibody (1:1000, Jackson Immunoresearch). The visual data were scanned using a fluorescence microscope.