Immunohistochemical Analysis of Mouse Developmental Disorder

Antibodies against CD31 and VEGFR2 were purchased from Abcam (Cambridge, UK). Immunohistochemical staining procedures were performed by Pfizer (Groton, CT) and at the Brigham & Women’s Hospital (Boston, MA). E14.5 has been established as the optimal time point for mouse developmental disorder phenotype analyses^{18 (link)}. In short, tissues were collected immediately after E14.5 embryo collection, fixed in 10% buffered formalin and embedded in paraffin. Four micrometer serial sections were cut and mounted on glass slides. One section from each lesion was stained with hematoxylin and eosin (H&E). Other slices were stained for CD31 (ab28364) or VEGFR2 (ab2349) with hematoxylin counterstaining following Abcam’s protocols.