Formation of fibronectin-matrix scaffolds in proangiogenic astrocytes. (A) Triple immunostaining (top) for fibronectin (FN, green), PECAM-1 (red), and PDGFRα (blue) and double immunostaining (middle and bottom) for lectin (red) and integrin α5 or β1 (green) at the sprouting vascular edges of P5 retina. (B and C) Retinal vessels 24 (B) or 48 (C) hours after intraocular injections of control IgG (left) or anti-integrin β1 antibody (right) at P2. (B) Immunostaining for PECAM-1 (red) and PDGFRα (green) at the sprouting vascular edges. Arrows indicate endothelial cells dissociating from astrocyte (AC) scaffolds. (C) PECAM-1 staining. (D) Immunostaining for PDGFRα (red) and fibronectin (green) in P7 retina. Fibronectin is expressed around blood vessels (arrowheads) but almost absent in astrocyte networks, which are not associated with blood vessels (arrows). (E) Flow cytometric analysis of retinal cells from Tie2-GFP transgenic mice at P7. Endothelial cells and astrocytes were sorted as GFP⁺ (0.26%) and PDGFRα⁺ (0.43%) fractions, respectively. (F) RT-PCR from total RNA of GFP⁺ and PDGFRα⁺ cells sorted by FACS. Fibronectin mRNA is expressed in both endothelial cells and astrocytes. (G) Red immunostaining for PDGFRα (left), ISH for fibronectin (middle), or VEGF (right) in P4 retinas. Arrowheads indicate the growing edge of the retinal vasculature depicted by immunostaining for type IV collagen (coll IV; green). Lower panels are the magnified views of growing vascular edges. Magnification, ×200 (A and D), ×400 (B), ×70 (C), ×63 (G, upper), ×252 (G, lower).