Hypoxia-induced deposition of collagen IV and fibronectin in endothelial ECM. (A) Whole mount immunofluorescence of P5 mouse retina was performed for detection of collagen IV (Col IV) and fibronectin (Fn). ECs were stained with isolectin B4 (IB4). Images correspond to the projection of 6 confocal sections at the vascular front. Arrows indicate sites of deposition of collagen IV and fibronectin by tip cells in the basement membrane. Arrowheads indicate the fibronectin network in the avascular zone. Bar: 20 μm. (B) HUVECs were cultured in normoxia or hypoxia for 5 days. Immunofluorescence of collagen IV (left panel) and fibronectin (right panel) was performed on either whole cell layer (cells) or ECM after detaching cells (ECM). Nuclei were stained with DAPI. Bar: 30 μm. See also supplemental Figure 1.