Fitc conjugated anti rat secondary antibodies

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).

VEGFR2-targeted UCA was incubated with FITC-conjugated anti-rat secondary antibodies (eBioscience, San Diego, CA) at room temperature for 30 min. After rinse and removing the free secondary antibodies, FITC-labeled targeted UCA was acquired. The antibody conjugation efficiency of VEGFR2-targeted UCA was detected according to fluorescent intensity of FITC-conjugated secondary antibodies. Particle size, size distribution and concentration of UCA were measured with the Accusizer 780 Optical Particle Sizer (Particle Sizing Systems, Santa Barbara, CA, USA). A drop of FITC-labeled targeted UCA suspension (about 50 μL in 1×10⁸ particles /mL) was applied to the microscope slide and examined under a fluorescent microscope (Olympus, Tokyo, Japan).