The microtubule tip complex proteins, EB1, p150^Glued, and Clasp1, are required for EC tubular morphogenesis in 3D collagen matrices. (A) ECs were treated with control (luciferase), p150^Glued, Clasp1, or EB1 siRNAs and suspended within 3D collagen matrices for 24 hours. Fixed cultures were stained with toluidine blue and photographed (upper panel). Bar equals 200 μm. Cultures were double stained with anticollagen antibodies to examine vascular guidance tunnel spaces that are created as a result of the tube formation (left lower panel) and anti-α tubulin antibodies to visualize the EC cytoskeleton under the indicated conditions (lower panel). Representative confocal microscopy images of control, EB1-, p150^Glued-, or Clasp1 siRNA–treated cells in 3D collagen matrices are shown (lower panel). Bar equals 20 μm. (B) Cultures from (A) were quantitated for EC lumen (B) and vascular guidance tunnel (C) formation. Data are shown as mean EC lumenal area ± standard deviation (n = 6) measured using Metamorph software. Statistical significance ***P < .0005.