Rat anti mouse cd31 primary antibody

Tissue immunofluorescent staining was performed to evaluate CD146 and CD31 expression in orthotopic tumor models according to established procedures.^{21 (link),22 (link)} The tumor was quickly removed after the terminal imaging time point (48 h) and prepared into slides at 5 μm. Frozen slides were fixed with cold acetone for 10 min and dried in air for 30 min. Tissue sections were then rinsed with PBS and blocked with 10% donkey serum at 25 °C for 60 min. Next, the slides were incubated with YY146 (10 μg/mL) and rat anti-mouse CD31 primary antibody (1:100, BD BioSciences) at 4 °C overnight. After rinsing with PBS, the slides were stained with AF488-labeled goat anti-mouse antibody and Cy3-labeled donkey anti-rat antibody. A hard mount medium (Vector Laboratories) was used to apply coverslips to each slide for fluorescence microscopy with DAPI. All images were acquired using an A1R confocal microscope (Nikon).

VEGF₁₂₁ was provided by GenScript Corp. (Piscataway, NJ). S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) was purchased from Macrocyclics, Inc. (Dallas, TX). Chelex 100 resin (50-100 mesh) and fluoresce in isothiocyanate (FITC) were purchased from Sigma-Aldrich (St. Louis, MO). Succinimidyl carboxymethyl PEG maleimide (SCM-PEG-Mal; molecular weight: 5 kDa; Creative PEGworks, Winston Salem, NC), rat anti-mouse CD31 primary antibody (BD Biosciences, San Diego, CA), AlexaFluor488- or Cy3-labeled secondary antibodies (Jackson Immunoresearch Laboratories, Inc., West Grove, CA), Bevacizumab (Avastin, Genentech, San Francisco, CA) and PD-10 desalting columns (GE Healthcare, Piscataway, NJ) were all acquired from commercial sources. Water and all buffers were of Millipore grade and pre-treated with Chelex 100 resin to ensure that the aqueous solution was free of heavy metal. All other reaction buffers and chemicals were obtained from Thermo Fisher Scientific (Fair Lawn, NJ).