Tie2-Cre;Nrp1^fl/– mutants contain tip cells that retain NRP1 expression, even though Tie2-Cre effectively activates the Rosa26^Yfp reporter in tissue macrophages as well as endothelial tip and stalk cells. (A-D) NRP1 (red) and IB4 (green) immunofluorescence staining of littermate E11.25 hindbrains lacking Cre or expressing a constitutively active Tie2-Cre transgene on an Nrp1^fl/– background; single NRP1 channels are shown below each panel (A′-D′). (B and D) Higher magnifications of the boxed areas in (A and C). Arrowheads indicate examples of tip cells, arrows show examples of tissue macrophages expressing NRP1. Clear arrows in (D) and asterisks in (D′) indicate the position of macrophages lacking NRP1, and curved arrows indicate endothelial stalk cells lacking NRP1. Scale bar represents 100 μm for (A,A′,C,C′). (E) An E11.5 hindbrain carrying a constitutively active Tie2-Cre transgene and the Rosa26^Yfp reporter was triple labeled for NRP1 (red), YFP (green), and IB4 (blue); single channels are shown in (E′-E′′′). Scale bar represents 50 μm.