Macrophages with activated Notch signaling localize to anastomosing sprouts and at vascular branchpoints. Whole mount P5 retinas. (A) F4/80 (red) and isolectin-B4 (blue) staining demonstrates localization of macrophages in close proximity to tip cells (arrowhead) and between neighboring sprouts (arrow). (B) Macrophages (green arrow) bridge between neighboring Dll4-postive (red) tip cells (arrowheads). Isolectin-B4 shown in blue. (C-F) Immunohistochemistry of retinas from Notch reporter mice expressing GFP downstream of a Notch-responsive promoter. Anti-GFP staining shown in green indicates activation of Notch signaling. Vessels were visualized with isolectin-B4 (blue). F4/80 staining shown in red. (C) Macrophages (red) with evidence of Notch signaling (arrows) localize between sprouts at the vascular front. (D) Proximally in the vascular plexus, macrophages with evidence of Notch signaling were found in close association with endothelial cells at vascular branchpoints (arrows). (E-F) Higher magnifications of the boxed areas in panels C and D. Arrowheads indicate anastomosing sprouts (E) or vascular branchpoints (F). Macrophages that were not in association with endothelial cells did not have evidence of Notch signal activation (indicated with stars). (G) The majority of TNR-positive macrophages in retinas were localized to vascular branchpoints (87.9% vs 12.07% of TNR-positive macrophages. (H-I) TNR-positive macrophages are overrepresented in the distal 20% of the retinal vasculature. Forty-two percent of all retinal macrophages were located in the distal 20% of the retinal vasculature. Of TNR-positive macrophages, 72.5% were located in the distal 20% of the retina. TNR-positive macrophages circled in white. Error bars represent data from n = 5 retinas from TNR mice. *P ≤ .05. Images in panels A through D shown at 40× original magnification, panel I shown at 4× original magnification. Data are representative of at least 3 independent experiments.