Fig. 2. VEGFA is required for arterial differentiation in limb skin. (A-F,H-M) triple immunofluorescence confocal microscopy using antibodies to either NRP1 (A-F, red, arrows and arrowhead) or CX40 (H-M, red, arrows and arrowhead), together with antibodies to PECAM1 (A-C,H-J, blue) and neurofilament (2H3) (A-C,H-J, green, open arrowheads). Arrowheads indicate large-diameter vessels. (G,N) Quantitative analysis of arterial marker expression in small-diameter vessels. Asterisks indicate statistically significant differences (P<0.05) in Vegf^flox/flox or Wnt1-Cre; Vegf^flox/flox compared with Wnt1-Cre; Isl1-Cre; Vegf^flox/flox (Student's t-test). (O-T) Whole-mount double labeling with TUNEL (O-T, red, arrows) and PECAM1 (O-Q, blue) reveals that no significant increase in apoptosis in the absence of nerve-VEGFA, confirmed by quantitative analysis (U). Scale bars: 100µm.