期刊
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
卷 -, 期 80, 页码 -出版社
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/50644
关键词
Neuroscience; Issue 80; Blood Cells; Endoderm; Motor Neurons; life sciences; animal models in situ hybridization; morpholino knockdown; progranulin; neuromast; proprotein convertase; anti-sense transcripts; intermediate cell mass; pronephric duct; somites
This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.
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