Blockade of pericyte recruitment to EC tubes leads to decreased basement membrane matrix deposition. EC/GFP-pericyte cocultures were established for 3 days and fixed for immunostaining analysis. Detergent-free immunostaining methods were used to assess the extracellular deposition of the key basement membrane components: collagen IV (Col IV), laminin (LM), and fibronectin (FN). (A) Quantification of immunostaining intensity is shown, demonstrating the decrease in basement membrane protein deposition under conditions of abrogated pericyte recruitment. (B) Intensity mapping of the representative images from panel C is shown to demonstrate the sites of highest basement membrane protein deposition, based on immunostaining patterns. (C) Representative images of the immunostained EC-pericyte cocultures are shown, with each of the basement membrane proteins in red and GFP-pericytes in green, under control versus PDGFRβ-Fc/EGFR-Fc–treated cultures. As shown by these images, there is decreased basement membrane protein deposition in cultures that have inhibited pericyte recruitment. The final column (CD31/Collagen IV) displays representative overlays of collagen IV (red), with the EC marker, CD31 (blue), and the corresponding pericytes (green) to show the relationship between the 3 structures. Bar equals 20 μm; n ≥ 5; P ≤ .01.