High-resolution in situ hybridization to whole-mount zebrafish embryos.
摘要：
The in situ hybridization (ISH) technique allows the sites of expression of particular genes to be detected. This protocol describes ISH of digoxigenin-labeled antisense RNA probes to whole-mount zebrafish embryos. In our method, PCR-amplified sequence of a gene of interest is used as a template for the synthesis of an antisense RNA probe, which is labeled with digoxigenin-linked nucleotides. Embryos are fixed and permeabilized before being soaked in the digoxigenin-labeled probe. We use conditions that favor specific hybridization to complementary mRNA sequences in the tissue(s) expressing the corresponding gene. After washing away excess probe, hybrids are detected by immunohistochemistry using an alkaline phosphatase-conjugated antibody against digoxigenin and a chromogenic substrate. The whole procedure takes only 3 days and, because ISH conditions are the same for each probe tested, allows high throughput analysis of zebrafish gene expression during embryogenesis.
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关键词：
Chorion Embryo, Nonmammalian Animals Zebrafish RNA Probes RNA, Messenger In Situ Hybridization Polymerase Chain Reaction Templates, Genetic Permeability
DOI：
10.1038/nprot.2007.514
被引量：
年份：
2008
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