Tip cells express secreted molecules that bind to stalk cells and are regulated by VEGF. (A-C) Whole mount ISH revealed tip cell expression (white arrows) of apelin (APLN), ESM1, and ANGPT2 (blue). (D) Whole mount ISH revealed expression of apj in stalk cells (red arrows). (E) ESM-1-AP fusion protein binds to stalk cells (red arrows). (F) Tie2 was detectable by antibody staining in stalk cells (red arrows) but not in tip cells. (G-L) Compared with PBS control injection (G-I), sFlt injection (J-L) leads to a decrease in APLN, ESM-1, and IGFBP3 expression. Vessels were stained with IsolectinB4 (green). The pictures represent 1 of 3 independent experiments with similar results. Scale bar 50 μm. Images of antibody stainings were acquired on a Leica TCS SP5 confocal microscope using a 63×/1.4 NA objective. Images of ISH were acquired on an Olympus BX50 microscope using a 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 and alkaline phosphatase signal by brightfield microscopy. Single channels were converted to grayscale in Photoshop CS2 and merged in ImageJ (ISH in blue).