Zebrafish whole mount high-resolution double fluorescent in situ hybridization.
Zebrafish whole mount high-resolution double fluorescent in situ hybridization.
复制标题
DOI：
10.3791/1229
复制
发表时间：
2009-03-25
期刊：
影响因子：
--
通讯作者：
Holley, Scott A
中科院分区：
文献类型：
--
作者：
Brend, Tim;Holley, Scott A
整体原位杂交是发育生物学中应用最广泛的技术之一。在此，我们介绍一种高分辨率的双重荧光原位杂交方案，用于分析单个基因的精确表达模式以及确定两个基因表达区域的重叠情况。该方案是使用碱性磷酸酶以及诸如NBT/BCIP和Fast Red等底物的标准原位杂交的改良版本（1,2）。此方案利用标准的地高辛和荧光素标记的探针以及酪胺信号放大（TSA）技术（3）。市售的TSA试剂盒允许灵活的实验设计，因为从绿色到远红色的荧光发射可与各种核染色剂（如碘化丙啶）或蛋白质的荧光免疫组织化学相结合使用。TSA产生一种反应性荧光底物，它能迅速与标记的反义核糖核酸探针附近的部分（通常是酪氨酸残基）共价结合。由此产生的染色模式具有高分辨率，通过激光扫描共聚焦显微镜可以观察到mRNA的亚细胞定位（3,4）。人们可以观察到染色体位点上的新生转录本，区分核染色和细胞质染色，并观察到其他模式，如mRNA的皮质定位。对果蝇的研究表明，大约70%的mRNA表现出特定的亚细胞定位模式，这些模式常常与编码蛋白质的功能相关（5）。当与计算机辅助的三维共聚焦数据集重建相结合时，我们的方案能够在整个脊椎动物胚胎中以亚细胞分辨率详细分析mRNA的分布。
Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red (1,2). This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) (3). The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy (3,4). One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein (5). When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.