Bx41f

The cells were seeded in a 6-well plate at a density of 5x10⁴ cells/well. The levels of intracellular ROS were measured with the fluorescent probe dichloro-dihydro-fluorescein diacetate (DCFH-DA; Beijing Baiaosentai Biotechnology). Following treatment with stimuli, the cells were incubated with DCFH-DA (10 µM) at 37˚C for 30 min. The cells and probe were mixed thoroughly for 30 min by inverting the flask once every 3-5 min. The cell suspension was centrifuged at 12,000 x g for 5 min at 4˚C, and the supernatant was discarded. The cells were resuspended and washed 3 times with 1 ml serum-free medium to remove the DCFH-DA that did not enter the cells. The cells were then centrifuged again at 12,000 x g for 5 min at 4˚C, and the supernatant was discarded, followed by the addition of 500 ml phosphate-buffered saline (PBS) to resuspend the cells. After 30 min, the cells were subjected to flow cytometry (using the parameters set for FITC) at an excitation wavelength of 535 nm and an emission wavelength of 610 nm to detect the fluorescence intensity before and after stimulation. The fluorescence images were obtained with fluorescence microscopy (BX41F, Olympus Corporation).

The tumor sections (6 μm) were fixed in 4% paraformaldehyde for 3–4 h at room temperature, and rinsed with phosphate-buffered saline (PBS). After washing with PBS, the non-specific binding sites were blocked with 10% goat serum (GTX27481, GeneTex) for 1 h at room temperature. The samples were incubated with one or two primary antibodies, that is, mouse monoclonal anti-CD34 (1:50, Abcam, USA) and rabbit polyclonal anti-α-SMA (1:50, Abcam, USA), simultaneously in 1% goat serum at 4°C overnight. Sections were washed with PBS and incubated in the dark for 1 h with secondary antibodies: Alexa Flour^® 568 goat anti-rabbit IgG (H+L) (1:200, A11011, Invitrogen) and Alexa Flour^® 488 goat anti-mouse IgG (H+L) (1:200, A1106, Invitrogen). After washing three times with PBS for 5 min, the nuclei were stained with DAPI (S36939, Invitrogen) for 15 min, and the sections were then examined under a confocal scanning microscope (BX41F; Olympus, Tokyo, Japan). The ratio of α-SMA/CD34 was calculated by dividing the positive area of α-SMA adjacent to CD34-positive vessels by the total area of CD34-positive tumor vasculature under five 200× high-powered randomly chosen fields per slide.