High-resolution in situ hybridization to whole-mount zebrafish embryos
High-resolution in situ hybridization to whole-mount zebrafish embryos
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DOI：
10.1038/nprot.2007.514
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Thisse, Bernard
中科院分区：
文献类型：
--
作者：
Thisse, Christine;Thisse, Bernard
原位杂交(ISH)技术可以检测特定基因的表达部位。该方案描述了地高辛标记的反义RNA探针对斑马鱼整体胚胎的ISH。在我们的方法中，以目的基因的PCR扩增序列为模板合成反义RNA探针，该探针标记有地高辛标记的核苷酸。胚胎在被地高辛标记的探针浸泡之前被固定和渗透。我们使用有利于特定杂交的条件来互补组织(S)中表达相应基因的mRNAs序列。洗掉多余的探针后，用碱性磷酸酶标记的地高辛标记抗体和显色底物进行免疫组织化学检测。整个过程只需要3天，因为每个被测探针的ISH条件都是相同的，所以可以高通量分析斑马鱼在胚胎发育期间的基因表达。
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.