Fig. 6. NRP1 is required for arteriogenesis in limb skin. Analysis of E15.5 limbs from Tie2-Cre; Nrp1^flox/- mutants or control littermates (Nrp1^flox/+) is shown. (A-D,F-I) Triple immunofluorescence confocal microscopy using antibodies to either CX40 (A-D, red) or SMA (F-I, red), together with anti-PECAM1 (A,B,F,G, blue) and neurofilament (2H3) (A,B,F,G, green). (E) Quantification of CX40 expression, and (J) of SMA⁺ cell coverage in small-diameter vessels. Asterisks indicate statistically significant differences (P<0.05) in Tie2-Cre; Nrp1^flox/- compared with Nrp1^flox/+ (Student's t-test). Nerve-blood vessel alignment in the Tie2-Cre; Nrp1^flox/- mutants appears normal (C versus D, H versus I, arrows and open arrowheads). Scale bars: 100 µm.