Alexa flour 488 goat anti mouse igg h l

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.