Diamidinophenylindole

The freshly harvested tissues were formaldehyde-fixed prior to freezing and were sectioned using a cryostat (Leika CM1850; Leica Microsystems GmbH, Nussloch, Germany) to 4 μm. The tissue sections were then incubated with rabbit anti-CD41 polyclonal antibody (1:100; ab63983), mouse anti-CD31 monoclonal antibody (1:100; ab24590) and/or mouse anti-VEGF monoclonal antibody (1:200; ab1316), which were all purchased from Abcam, overnight at 4°C. The tissue sections were then incubated with a secondary goat anti-rabbit fluorescein isothiocyanate (111-095-003; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) and goat anti-mouse Rhodamine (115-295-003; Jackson ImmunoResearch Laboratories, Inc.) at 37°C for 1 h. The nuclei were counterstained with diamidinophenylindole (Sigma-Aldrich, St. Louis, MO, USA) and fluorescent images were captured using microscopy (BX51; Olympus, Tokyo, Japan) and analyzed using Image-Pro Plus software for colocalization.