In vivo tumor angiogenesis

The protocols for animal experiments were reviewed and approved by the Ethical Committee on Animal Experiments of Animal Care Committee of Fudan University (Fudan, China). Male BALB/c nu/nu mice at 4–6 weeks of age weighing 18–20 g were obtained from SLAC Laboratory Animal Co., Ltd. Mice were housed in animal rooms with a 10-h light/14-h dark cycle and at a constant temperature (22–27°C). Animals had free access to standard rodent chow and water. Following exposure to acute or intermittent hypoxia, liver cancer cells (2×107) mixed with endothelial cells (5×106) were injected subcutaneously into the flanks of the mice (each group, n=3) for the evaluation of tumor angiogenesis. The immunohistologic staining of endothelial cells was subsequently performed. Briefly, subcutaneous tumors were fixed in 10% formaldehyde solution for 10 h at room temperature, embedded in paraffin and cut into 5 µm thick slices. Some slices were stained with hematoxylin and eosin (H&E; Beyotime Institute of Biotechnology) according to a standard procedure (17). For VEGF and CD31 detection, tissue slices were deparaffinized in xylene for 20 min at room temperature, rehydrated in gradient ethanol (100, 95, 90 and 80%) and treated with 0.3% H2O2 for 15 min at room temperature to block endogenous peroxidase activity. To retrieve the antigen, slices were heated in 10 mM citrate buffer (pH 6.0) in a microwave oven at 95°C three times for 5 min. Slices were then blocked with 20% goat serum (Beyotime Institute of Biotechnology) for 30 min at room temperature, and were incubated with VEGF antibody (1:100; cat. no. ab51745; Abcam) or CD31 antibody (1:50; cat. no. ab28364; Abcam) at 4°C overnight and with the secondary antibody (1:50; cat. no. A0208, Beyotime Institute of Biotechnology) at room temperature for 30 min. Slices were treated with 3,3′-diaminobenzidine tetrahydrochloride (DAB; Beyotime Institute of Biotechnology), counterstained with hematoxylin and visualized under an Olympus IX73 inverted microscope. The cellular brown staining was considered as positive reaction.