Tetralogy of Fallot and Alterations in Vascular Endothelial Growth ...
Tetralogy of Fallot and Alterations in Vascular Endothelial Growth ...
Tetralogy of Fallot and Alterations in Vascular Endothelial Growth ...
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Ms # CIRCRESAHA/2006/140772-onl<strong>in</strong>e supplement/R2<br />
Onl<strong>in</strong>e Supplement<br />
<strong>Tetralogy</strong> <strong>of</strong> <strong>Fallot</strong> Correlates with <strong>Alterations</strong> <strong>in</strong> Notch-signall<strong>in</strong>g <strong>in</strong> Mouse<br />
Embryos Solely Express<strong>in</strong>g the VEGF120 Is<strong>of</strong>orm<br />
Nynke M.S. van den Akker, Daniël G.M. Mol<strong>in</strong>, Patricia P.W.M. Peters, Saskia Maas,<br />
Lambertus J. Wisse, Ronald van Brempt, Conny J. van Munsteren, Margot M.<br />
Bartel<strong>in</strong>gs, Robert E. Poelmann, Peter Carmeliet, Adriana C. Gittenberger-de Groot<br />
Mouse embryos <strong>and</strong> tissue process<strong>in</strong>g<br />
Materials <strong>and</strong> Methods<br />
All animal experiments were approved by the Animal Ethics Committee <strong>of</strong> the Leiden<br />
University <strong>and</strong> performed accord<strong>in</strong>g to the Guide for the Care <strong>and</strong> Use <strong>of</strong> Laboratory<br />
Animals published by the NIH. Vegf+/120 mice were crossed to obta<strong>in</strong> Vegf120/120<br />
embryos <strong>and</strong> Vegf+/+ wild type littermates. The morn<strong>in</strong>g <strong>of</strong> the vag<strong>in</strong>al plug was stated<br />
embryonic day (E)0.5. Pregnant females were sacrificed by cervical dislocation. The<br />
embryos were isolated <strong>and</strong> either snap-frozen <strong>in</strong> liquid nitrogen or fixed <strong>in</strong> 4%<br />
paraformaldehyde/phosphate-buffered sal<strong>in</strong>e (0.1 Mol/L, pH7.4), dehydrated <strong>and</strong><br />
embedded <strong>in</strong> paraff<strong>in</strong>. In total, 34 Vegf+/+ <strong>and</strong> 47 Vegf120/120 embryos rang<strong>in</strong>g from<br />
E10.5 to E19.5 were embedded <strong>in</strong> paraff<strong>in</strong> <strong>and</strong> used for microscopic analyses. Of the<br />
snap-frozen embryos (12 Vegf+/+ <strong>and</strong> 13 Vegf120/120 embryos <strong>of</strong> E14.5, E16.5 or<br />
E18.5) hearts were dissected <strong>and</strong> ve<strong>in</strong>s <strong>and</strong> great arteries were removed <strong>and</strong> stored <strong>in</strong><br />
RNAlaterice (Ambion, Cambridgeshire, UK) at -80 o C before use for RT-qPCR.<br />
Immunohistochemistry<br />
Downloaded from<br />
http://circres.ahajournals.org/ by guest on February 7, 2013<br />
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