Figure 7. Reduced vessel integrity in the Eogt−/− retina.
(A) Immunostaining with fibrinogen (green) and α-SMA (magenta) antibodies in P15 wild-type, Eogt−/−, Tek-Cre, Tek-Cre:EogtF/F, Notch1+/−, and Rbpj+/− retinas. Arrows indicate fibrinogen staining outside vessels stained by IB4 (white). Three-dimensional images were constructed from confocal images by maximum intensity projection. (B) Higher magnification three-dimensional images of Eogt−/− retina constructed from confocal images using the Alpha-blend method. Below, single channel images showing fibrinogen (green) and IB4 (white) staining. (C) Sulfo-NHS-LC-biotin was perfused into P15 wild-type and Eogt−/− mice and extravasation determined immediately after perfusion by staining with CF488A-conjugated streptavidin (green) and Dylight594-conjugated IB4 (white). Three-dimensional reconstructions were created by maximum intensity projection. Enlarged images of boxed area are shown (right). (D) Sulfo-NHS-LC-biotin was perfused into P15 wild-type, Eogt−/−, Notch1+/−, Eogt−/−Notch1+/− mice as in (C). Quantification of the number of extravasation sites in 210 × 210 μm squares (n = 6 per retina per mouse) is shown. Note that sulfo-NHS-LC-biotin extravasation in Eogt−/− retina is augmented in compound mutant mice. Data represent mean ± standard error; p values determined by Welch's t test. ***p≤0.001. (E) Whole-mount images of wild-type or Eogt−/− P15 retinas stained with IB4 (cyan) and anti-αSMA (magenta) antibody. (F) Whole-mount staining of wild-type and Eogt−/− P15 retinas using IB4 (white) together with anti-fibrinogen (green) and anti-NG2 (magenta) antibodies.
DOI: http://dx.doi.org/10.7554/eLife.24419.018
Figure 7—figure supplement 1. Sulfo-NHS-LC-biotin was perfused into P15 wild-type, Eogt−/−, Tek-Cre, Tek-Cre;EogtF/F, Notch1+/−, Rbpj+/−, Eogt−/−Notch1+/−, Eogt−/−Rbpj+/− mice and stained with CF488A-conjugated streptavidin (green) and Dylight594-conjugated IB4 (white) immediately after perfusion.
