Extracellular deposition of basement membrane components is observed only when EC are cocultured with pericytes during EC-pericyte tube coassembly. ECs were cultured alone or with pericytes and allowed to randomly assemble in a 3D collagen for 5 days, fixed, and processed for immunofluorescence (HUVECs-bovine pericytes). Staining was done without detergent to assure extracellular staining only. (A) Basement membrane matrices were stained for the following: collagen IV, laminin, nidogens 1 and 2, perlecan, and fibronectin (red staining), and GFP pericytes (green staining). EC-only cultures show very little extracellular deposition of the indicated molecules. EC-pericyte cocultures show a dramatic increase in extracellular deposition of the basement membrane components along with fibronectin. (B) α-Tubulin staining was conducted as a methods control. (C) Pericytes are localized within vascular guidance tunnels; EC-GFP pericyte cocultures in 3D collagen gels were allowed to coassemble, and then stained with an anti-collagen type I antibody. Bar equals 25 μm. (D) Separate gels from the same cultures were then immunostained with an anti-laminin antibody that shows localized laminin staining between the 2 cell types. Bar equals 25 μm. (E-F) Electron microscopy was performed, and representative images are shown revealing the deposition of basement membrane matrix material (arrowheads) between ECs and pericytes, corresponding to the position of anti-laminin staining shown in (D); this deposition and organization is not seen when ECs are cultured alone (F). Bar equals 2 μm.