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7144255884
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note
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For whole mount in situ hybridization, E8.5 to E10 embryos were fixed and processed according to standard protocols. For radioactive in situ hybridization, E11.5 to E12.5 embryos were isolated and processed as for histological analysis. Hybridization was performed according to standard protocols. The FADD probes used were a 1.3-kb full-length murine FADD cDNA and a 0.8-kb 3′ untranslated region fragment.
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7144260737
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7144258771
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note
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To examine the development of the vascular endothelium, a mutation in the flk1 gene marked with the lacZ gene (23) was bred to heterozygocity into the FADD mutant background. β-Gal staining was performed according to standard protocols.
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7144228292
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BrdU (100 μg per gram of body weight) was injected intraperitoneally into pregnant females at E10.5 to E11.5. Females were killed 1 hour after injection, and the embryos were dissected and fixed in 70% ethanol for 2 hours at 4°C, and then processed as for H&E staining. Sections were incubated with a monoclonal antibody to BrdU (Beckton Dickinson) at a 1:10 dilution. Staining was performed as described (24), with a biotinylated secondary antibody against mouse immunoglobulin G and avidin-conjugated peroxidase (Vector Laboratory).
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+/- ES cell clones and transferred them to CD1 pseudopregnant foster mothers. Chimeric embryos were dissected between E10.5 and E11.5 and stained for β-Gal activity.
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We thank V. M. Dixit for DR3 and DR4 vectors and P. H. Krammer for agonistic antibody to Fas; H. Hsu, L.-M. Boucher, and members of T.W.M.'s laboratory for material and instructive discussions; I. del Barco Barrantes and B. Hum for technical assistance; M. Saunders for scientific editing; and I. Ng for administrative support. Supported in part by a grant from the National Cancer Institute of Canada (NCIC) and grant CA13106 from the National Cancer Institute and Medical Research Council of Canada.
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