(A–F) ISH for VEGF combined with IHC for collagen IV. Note the increase of VEGF-expressing cells in Lif^–/– mice in the vascularized area. Boxed regions in B and E are shown at higher magnification in C and F as indicated. Dotted lines in A, B, D, and E indicate the sprouting edges. (G–J) Triple IHC for PDGFRα, PECAM-1, and HIF-1α (G and I) or for PDGFRα, HIF-1α, and DAPI (H and J) in P4 retinas of Lif^+/+ and Lif^–/– mice. Note the increased number of astrocytes with HIF-1α (arrows) and nuclear translocated HIF-1α proteins (arrowheads) in the vascularized area of Lif^–/– retinas. (K) Quantitative RT-PCR of vegfa with isolated RNA from various retinal stages (n = 5 per group). (L) Quantification of astrocytes with HIF-1α in the vascularized area (average numbers from 8 random FOV in the vascularized area per retina; n = 6). (M) Quantitative PCR of hif-1α for isolated RNA from P4 retina (n = 5). (N–S) ISH for VEGF combined with isolectin staining in P4 trachea of Lif^+/+ and Lif^–/– mice. Note the increase in VEGF-expressing mucosal epithelial cells in Lif^–/– mice, especially in the vascularized area. Dotted lines flank a cartilaginous ring area. (T–Y) Double IHC of PECAM-1 and VEGF on E11 embryos. (Z and AA) Quantitative PCR of vegfa for isolated RNA from P4 trachea (Z) or E11 embryos (AA) (n = 5 per group). Scale bars: 50 μm. *P < 0.03 versus Lif^+/+.