Computational protocol: VE-PTP regulates VEGFR2 activity in stalk cells to establish endothelial cell polarity and lumen formation

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Protocol publication

[…] Analyses repeated three times and representative results are shown. Quantification of immunoblotting was done using ImageJ (NIH). For sequential immunoprecipitation, the first precipitate with anti-FLAG antibody was eluted from protein G-Sepharose beads with 0.1 M glycine-HCl, pH 3.4, neutralized to pH 7.4, followed by the second immunoprecipitation with anti-V5 antibody; the supernatant was immunoprecipitated with anti-VEGFR2 antibodies. Samples were processed further by immunoblotting as indicated. [...] Whole-mount antibody staining of zebrafish embryos with anti-ZO1 (Sigma), anti-Pdxl and anti-Cdh5 antibodies was performed using acetone permeabilization. For staining with the zebrafish anti-VE-Cadherin/Cdh5 antibody, embryos were fixed in 2% PFA/PBS/0.1% Tween (PBST), permeabilized in PBST/0.5% Triton X-100, and incubated in primary antibody-diluted 1:500 in PBST/5% normal goat serum/1% BSA/0.1% Triton X-100. Embryos were washed in PBST and stained with goat anti-rabbit or goat anti-mouse Alexa-568 secondary antibodies (Invitrogen). Images were collected on a Zeiss LSM 700 confocal microscope. Image analysis was done using ImageJ (NIH) and Imaris (Bitplane). […]

Pipeline specifications

Software tools ImageJ, Imaris
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Danio rerio, Mus musculus
Diseases Teratoma