Alexa647 conjugated streptavidin

We performed the proliferation analysis using the Click-iT EdU Alexa Fluor 488 Imaging Kit (Life Technologies, C10337). P3 or P7 pups were injected with 300 μg of EdU (5 mg/ml) and were killed 2 h later. We stained whole-mount retinas with anti–Erg-1/2/3 and IB4 biotin conjugate followed by Cy3-donkey anti-rabbit, 1/200 (Jackson ImmunoResearch, 711.165.152) and Alexa 647–conjugated streptavidin, 2 μg/μl (Jackson ImmunoResearch, 016-600-084). EdU staining was done according to the manufacturer's protocol. We imaged the retinas with a Nanozoomer slide scanner. EdU⁺ and Erg-1/2/3⁺ double-labeled nuclei were counted as proliferating endothelial cells. One 45° wedge was quantified for each retina. Double-labeled cells were counted in a 250-μm-wide region localized at the angiogenic front to calculate the percentage of proliferating endothelial cells.
The xCELLigence RTCA DP analyzer was used to measure the proliferation of control and ROBO1- and ROBO2-knockdown HUVECs in response to complete ECGM-2 medium (Lonza) or Slit2 (6 nM, R&D Systems). E-16 tissue culture plates (ACEA Biosciences) coated with 0.1% gelatin were seeded with 5,000 cells per well. The plates were monitored every 15 min for 48 h. For each condition, at least three replicate wells were analyzed.