Del-1 interactions with αvβ5 regulate integrin αvβ3 expression. (a and b) Cryosections of CAMs stimulated with Del-1 were incubated with Ab’s against integrin αvβ3 or αvβ5 in combination with Ab’s against vWF. Integrin expression was detected with a rhodamine-conjugated secondary Ab and vWF with an FITC-conjugated secondary Ab. Integrin expression on blood vessels is seen as orange-yellow structures. Representative sections were photographed at ×200. White bar indicates 10 μm. (b) Average integrin expression levels for each integrin in vessels were quantified by determining the average number of red pixels in vessels that was greater than background and dividing by the average number of green pixels greater than background. (c) Integrins αvβ3 and αvβ5 were immunoprecipitated from lysate CAMs stimulated with bFGF or Del-1. Immunoprecipitates were immunoblotted with Ab’s against the cytoplasmic tails of the β3 and β5 subunits. Ratios of αvβ3 to αvβ5 were determined by densitometry. (d) CAMs were stimulated with Del-1 in the presence or absence of saline, anti-αvβ5, or W6/32 control isotype-matched Ab’s. Cryosections of CAMs were then incubated in anti-αvβ3 and anti-vWF Ab’s. Integrin αvβ3 expression was detected with a rhodamine-conjugated secondary Ab and vWF with an FITC-conjugated secondary Ab. Representative sections were photographed at ×200. White bar indicates 10 μm. Integrin αvβ3 expression on blood vessels is seen in yellow. Blood vessels are indicated with arrows. cIgG, control IgG. (e) Expression levels of integrin αvβ3 in vessels were quantified as in b.