Computational protocol: The VEGF receptor Flt-1 spatially modulates Flk-1 signaling and blood vessel branching

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Protocol publication

[…] Day 8 ES cell cultures were rinsed with PBS and fixed for 5 min in ice-cold methanol-acetone (50:50) for PECAM or β-galactosidase staining or 4% PFA in PBS for Flk-1 staining. For PECAM staining, fixed cultures were reacted with rat anti–mouse PECAM at 1:1,000 (MEC 13.3; BD Biosciences) and donkey anti–rat IgG (IgG; H+L) TRITC at 1:100 (Jackson ImmunoResearch Laboratories) or goat anti–rat conjugated to AlexaFluor488 (IgG; H+L) at 1:200 (Invitrogen) as described previously (). For β-galactosidase staining, cultures were reacted with rabbit polyclonal anti–β-galactosidase at 1:300 (Cappel Laboratories) and donkey anti–rabbit IgG (IgG; H+L) TRITC at 1:100 (Jackson ImmunoResearch Laboratories). For Flk-1 and pFlk (Tyr-1173/1175) staining, cultures were blocked in staining medium (5% goat serum in PBS) for 1 h at 37°C, and all antibodies were diluted into staining medium. Cultures were incubated in phospho–VEGFR-2 (Tyr-1175) rabbit antibody (19A10; Cell Signaling Technology) at 1:200 overnight at 4°C and after PBS washes were incubated with goat anti–rabbit IgG conjugated to AlexaFluor488 (Invitrogen) at 1:400 for 2 h at RT. Cultures were then incubated with rat anti–mouse Flk-1 antibody (BD Biosciences) at 1:200 overnight at 4°C and with goat anti–rat IgG conjugated to AlexaFluor568 (Invitrogen) for 1 h at RT and rinsed in PBS. PECAM-stained cultures were viewed and photographed with an inverted microscope (IX-50; Olympus) outfitted with epifluorescence using a 10× NA 0.25 CPlan RT objective (Olympus) and a camera (DP71; Olympus) with DP Controller version 3.1.1.267 software (Olympus). Flk-1– and β-galactosidase–stained cultures were analyzed with a confocal microscope (LSM 5 PASCAL; Carl Zeiss, Inc.) using either a 40× NA 1.3 EC Plan-Neofluor oil objective (Carl Zeiss, Inc.) or a 100× NA 1.4 plan-Apochromat oil objective (Carl Zeiss, Inc.) at RT using PASCAL Release version 4.2 SP1 acquisition software (Carl Zeiss, Inc.). For the β-galactosidase–stained cultures, ∼10 confocal images were acquired through 12 μm of thickness on the z axis and were combined and flattened. Minor adjustments (brightness and contrast to the whole panel) were done using Photoshop CS2 (Adobe).To quantify the vascular area labeled with PECAM antibody, PECAM-stained cultures were photographed and analyzed as described previously (). In brief, four to six wells were analyzed for each genotype. For each well, six to eight images were acquired sequentially for analysis. Percent PECAM area means for each well were calculated, and the mean of four wells for each clone was used to determine SD values. Branch point analysis was performed on similar images from PECAM-stained cultures as described previously (). The mean branch point score from 8–12 pictures for each clone was used to determine SD values. All values were statistically analyzed using the two-tailed t test. Flk-1–stained cultures were analyzed by counting the number of total Flk-1–positive cells that also were positive for pFlk (Tyr-1173/1175) in representative areas. Quantitative analysis of the ratio of pFlk (Tyr-1173/1175) to total Flk staining was performed by outlining individual endothelial cells and using MetaMorph software (MDS Analytical Technologies) to calculate the ratio. [...] Western blot analysis was performed as described previously with some modifications (). In brief, day 8 ES cell cultures were lysed into radioimmunoprecipitation assay buffer supplemented with protease inhibitors. Lysates were centrifuged at 12,000 g for 10 min, and supernatants were separated on an 8% SDS-polyacrylamide gel. Gel transfer was to a polyvinylidene fluoride membrane (GE Healthcare) under standard conditions. The phospho-Flk signal was detected by incubation with antiphospho-VEGFR2 (Tyr-1175; 1:500; Cell Signaling Technology) and HRP-labeled anti–mouse secondary antibody (1:5,000; GE Healthcare). Total Flk-1 was detected by using rat ant–mouse Flk-1 antibody (1:500; BD Biosciences) and HRP-labeled anti–rat secondary antibody (1:5,000; GE Healthcare). After detection by enhanced chemiluminescence (GE Healthcare), the results were quantified by densitometry using ImageJ (National Institutes of Health). […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Diseases Gingival Overgrowth