Tip cells express basement membrane components. (A-B) Whole mount ISH (blue) confirmed enhanced Nidogen-2 (nid2) expression in tip cells of wild-type mice (white arrows) and a broader expression in DLL4^+/− mice (C-D). (E-F) Nidogen-1 (nid1) mRNA (blue) is expressed in the vascular front of wild-type mice, including tip cells (white arrows) and stalk cells (red arrows). (G-H) Nidogen-1 mRNA is up-regulated in DLL4^+/− mice. (I-L) Whole mount immunohistochemistry revealed Nidogen-1 protein (red) in the basement membrane of all retinal vessels in wild-type mice, at the exclusion of filopodia (K-L). IsolectinB4 was used to counterstain vessels (green). The pictures represent 1 of 3 independent experiments with similar results. Scale bars: (A,C,E,G,I,J) 250 μm; (B,D,F,H) 50 μm; (K-L) 40 μm. Images of antibody stainings were acquired on a Leica TCS SP5 confocal microscope using a 10×/0.3 or 63×/1.4 NA objective. Images of ISH were acquired on an Olympus BX50 microscope using a 4×/0.16 NA or 20×/0.05 NA objective and captured using a Coolsnap camera through IPLab software version 3.2.4. Vessels were visualized with IsolectinB4 directly conjugated to AlexaFluor488 and the ISH signal by bright-field microscopy. Single channels were converted to grayscale in Photoshop CS2 and merged in ImageJ (ISH in blue).