Dylight 594 labeled gsl 1 isolectin b4

The murine aortic ring angiogenesis assay was set-up as previously described9 (link). In some experiments, aortic rings were infected with adenoviral constructs overnight in Optimem (Life Technologies) on the day of harvest. Medium containing either no addition (control), or supplemented with 30 ng/ml VEGF, was replaced and fresh growth factors supplemented every 2-3 days. After 1 week, the aortic rings were fixed with 4% formalin for 30 minutes at room temperature. The fixed rings were permeabilised with PBS containing 0.25% Triton X-100 for 15 minutes (2 times), blocked with casein solution for 30 minutes and incubated overnight with DyLight 594 Labeled GSL I - isolectin B4 (Vector Labs, 1:100) and anti-neuropilin 1 antibody (Abcam, clone EPR3113) at 4°C. Negative controls were sections that were either incubated with PBS (for the isolectin B4 - DyLight 594 antibody conjugate control) or rabbit IgG (for the Nrp1 antibody control) instead of primary antibody. Next morning, rings were washed three times with PBS, and incubated for 1 h in the dark with Alexa Fluor® 488-conjugated donkey anti-rabbit IgG (Life Technologies), mounted on slides and imaged under a fluorescent microscope (Zeiss Axio Imager A1). The isolectin stained images were analysed using Image J to evaluate the total area of outgrowth and number of branch points by an observer blinded to the treatments given.