Rat anti mouse cd31 antibody

Tumor masses or ‘early EPCs’ were fixed with 4% paraformaldehyde (PFA) in PBS. Sections were prepared from OCT embedded tumor tissues and washed in PBS. After blocking with 1% bovine serum albumin (BSA, Sigma) in PBS, sections were stained with rat anti-mouse CD31 antibody overnight at 4°C, and then with Alexa Flour 647 -conjugated anti-rat antibody (Invitrogen). Fixed cells were stained with FITC-conjugated anti-CD34 monoclonal antibody (GeneTex) and PE-conjugated anti-VEGFR2 monoclonal antibody (BD Biosciences).

After ultrasound imaging, the mice were sacrificed immediately and uterus were harvested for the histological study. Double staining for VEGFR2 and CD31 was performed to confirm co-localization of VEGFR2 on CD31-positive vascular endothelial cells. Briefly, the dissected uterus samples were covered with Tissue-Tek (Sakura), and then frozen in liquid nitrogen vapor. The tissue sections (10 μm) were cut with a cryostat microtome (CM1950; Leica, Heidelberg, Germany) and fixed with pre-cooled acetone for 2 min, followed by drying in air for at least 1 h. Then, the sections were rinsed with PBS for 5 min and incubated with 0.03% H₂O₂ in PBS, and subsequently blocked with 5% goat serum for 1 h at room temperature. After that, slides were co-incubated with rabbit anti-mouse VEGFR2 antibody (Cell Signaling Technology Inc., Danvers, MA) and rat anti-mouse CD31 antibody (eBioscience, San Diego, CA) at a dilution of 1:200 overnight at 4°C. Cy3-conjugated anti-rabbit (biorbyt, Cambridge, UK) and FITC-conjugated anti-rat secondary antibodies (eBioscience, San Diego, CA) were used to visualize the expressions of VEGFR2 and CD31, respectively. Fluorescent images were acquired at × 200 magnifications with a laser scanning confocal microscope (TCS SP5, Leica, Germany).