Antibody against cd31

Tissue was harvested and embedded in OCT (Sakura Finetek USA, Inc., Torrance, CA). 10 μm thick frozen sections were fixed in acetone and immunostained using an antibody against CD31 (1:200, Abcam, Cambridge, MA). Nuclei were stained with DAPI. Randomized, duplicate microvessel counts per high power field (400×) were conducted on each skin or intra-implant sample following CD31 staining. To avoid sampling error, microvessels were defined as distinct areas of positive staining having a length to width ratio greater than or equal to one, and in close association with nuclei.

Aortic roots were first incubated with the antibody against CD31 (Abcam, 1:50) for 4 h followed by incubation with donkey anti-rabbit IgG labeled with Alexa Fluor 594 (Invitrogen, 1:1000) for 30 min at room temperature under dark conditions. Treated HUVECs on glass coverslips placed in six-well plates were fixed with paraformaldehyde (4%, 20 min). After permeabilization with Triton X-100 (0.1%, 2 min), the aortic roots and cells were incubated with the TUNEL reaction mixture (Fluorescein for tissues and TMR red for cells) in the dark (1 h) and then stained with DAPI. The images of the samples were captured by an Olympus BX53 fluorescence microscope (Tokyo, Japan) and the percentage of TUNEL or CD31-TUNEL positive cells was measured using the Image-Pro Plus software (version 6.0, Media Cybernetics, LP, USA). The endothelial cells with DNA damage were calculated as the percentage of the number of TUNEL-positive cells to the total HUVECs or CD31-TUNEL positive cells to the plaque areas in the aortic roots.

Human glioma cell lines U87 and U251 (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM supplemented with 2 mmol/L l-glutamine, 10% fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin, 5% CO2 at 37°C. Antibodies against GSK-3β, phospho-GSK-3β (Ser9), β-catenin, AKT, phospho-AKT (Ser473), p70S6K1, phospho-p70S6K1, mTOR, phospho-mTOR, β-Tubulin were obtained from Cell Signaling (Beverly, MA, USA). HIF-1α was from BD Biosciences (Franklin Lakes, NJ, USA), and GAPDH was purchased from KangCheng Biotech (Shanghai, China), while antibody against CD31 was supplied by Abcam (Cambridge, UK).