Notch1 in macrophages is important for macrophage localization during retinal angiogenesis. Whole mount P5 retinas were stained by immunohistochemistry for F4/80 or isolectin-B4 to visualize macrophages or endothelial cells, respectively. (A) Macrophages (red) in control retinas were found in close proximity to endothelial cells (green) and at vascular branchpoints (arrows). (B) F4/80 staining of image shown in panel A. Macrophages in control retinas were densely localized to the vascular front, at the leading edge of vessel migration and anastomosis. (C) Macrophages (red) in retinas from Notch1^+/− mice were often not associated with vascular branchpoints (indicated with stars). (D) F4/80 staining of image shown in panel C. Macrophages in Notch1^+/− retinas were scattered at the vascular front where vessel anastomosis occurs. (E-G) Retinal angiogenesis in mice with myeloid-specific reduction of Notch1 was characterized by an increased frequency of elongated sprouts that failed to anastomose with neighboring sprouts. For quantification of elongated sprouts, 10× images of retinas were used to assess the frequency of sprouts longer than 100 μm, normalized to the total number of sprouts in that field. Sprouts were defined as endothelial protrusions from the vascular front that did not branch. Results were averaged for each genotype. Error bars represent SEM of n = 4 control, n = 6 LysMCre; Notch1^flox/+, and n = 4 LysMCre;Notch1^flox/flox retinas. (H-L) Decreased macrophages at the retinal vascular front in mice with myeloid-specific loss of Notch1. For quantification, paneled 30× images of the entire retina were taken. To determine F4/80 staining density (shown in red), the number of red pixels within the outer 20% of the retina (indicated by white line in panels H and I, outline of vascular plexus shown in white) was counted and compared with the total area covered by vasculature within that region. The leading edge was defined as the distal 20% of vasculature measured from the center of the optic nerve to the edge of the vascular front. Error bars represent SEM of n = 4 control and n = 4 LysMCre;Notch1^flox/flox retinas. *P ≤ .05. (K-L) Macrophages in control retinas were found at the vascular front and localized to vascular branchpoints (arrowheads), whereas there was decreased macrophage density at the vascular front in retinas from mice with loss of Notch1 in the myeloid lineage (arrows). Images shown at 40× (A-D,K,L), or 10× (E,F,H,I), original magnification. Images are representative of at least 3 independent experiments.