Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems.
Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems.
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DOI：
10.1186/1471-213x-11-43
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发表时间：
2011-07-04
影响因子：
--
通讯作者：
Hauptmann G
中科院分区：
文献类型：
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作者：
Lauter G;Söll I;Hauptmann G
整体原位杂交（WISH）被广泛用于表征发育和成人大脑和其他组织中的基因表达模式。为了了解一个新的基因是否可能参与一个独特的脑亚部、核或神经元谱系的特化，将其表达与已知的区域或神经元标记基因的表达相关联通常是有用的。双色荧光原位杂交（FISH）可用于在细胞分辨率下比较不同的转录本分布。常规的双色FISH方案需要两轮单独的基于辣根过氧化物酶（POD）的转录物检测，其涉及酪胺信号放大（TSA）和在第二轮检测之前第一次应用的抗体-酶缀合物的失活。我们在这里表明，碱性磷酸酶（AP）底物固红和固蓝可用于显色以及荧光可视化的成绩单。为了实现高信号强度，我们通过过氧化氢处理优化了胚胎透化特性，并通过应用粘度增加聚合物硫酸葡聚糖优化了杂交条件。所获得的信号增强，使我们能够开发一个敏感的双色FISH协议相结合的AP和POD报告系统。我们表明，AP-坚牢蓝和POD-TSA-羧基荧光素（FAM）检测的组合提供了一个强大的工具，同时荧光可视化两个不同的成绩单在斑马鱼大脑。不同检测系统的应用允许一步抗体检测程序用于转录物的可视化，这显著减少了工作步骤和动手时间，将方案缩短了一天。第一个应用的报告酶的失活变得不必要，因此可以消除由于失活不足而导致的共定位的假阳性检测，这是常规双色FISH的问题。由于POD活性被底物过量相当快地淬灭，因此即使在应用TSA时，丰度较低的转录物通常也不能有效地可视化。使用AP-Fast Blue荧光检测可以为荧光转录物可视化提供有用的替代方案，因为AP反应可以以高信噪比进行延长的时间。因此，我们的协议提供了一种新的替代比较两种不同的基因表达模式在胚胎斑马鱼脑细胞水平。我们方法的原理是为在斑马鱼中使用而开发的，但可以很容易地包含在其他模式生物的整体安装FISH方案中。
Whole-mount in situ hybridization (WISH) is extensively used to characterize gene expression patterns in developing and adult brain and other tissues. To obtain an idea whether a novel gene might be involved in specification of a distinct brain subdivision, nucleus or neuronal lineage, it is often useful to correlate its expression with that of a known regional or neuronal marker gene. Two-color fluorescent in situ hybridization (FISH) can be used to compare different transcript distributions at cellular resolution. Conventional two-color FISH protocols require two separate rounds of horseradish peroxidase (POD)-based transcript detection, which involves tyramide signal amplification (TSA) and inactivation of the first applied antibody-enzyme conjugate before the second detection round. We show here that the alkaline phosphatase (AP) substrates Fast Red and Fast Blue can be used for chromogenic as well as fluorescent visualization of transcripts. To achieve high signal intensities we optimized embryo permeabilization properties by hydrogen peroxide treatment and hybridization conditions by application of the viscosity-increasing polymer dextran sulfate. The obtained signal enhancement allowed us to develop a sensitive two-color FISH protocol by combining AP and POD reporter systems. We show that the combination of AP-Fast Blue and POD-TSA-carboxyfluorescein (FAM) detection provides a powerful tool for simultaneous fluorescent visualization of two different transcripts in the zebrafish brain. The application of different detection systems allowed for a one-step antibody detection procedure for visualization of transcripts, which significantly reduced working steps and hands-on time shortening the protocol by one day. Inactivation of the first applied reporter enzyme became unnecessary, so that false-positive detection of co-localization by insufficient inactivation, a problem of conventional two-color FISH, could be eliminated. Since POD activity is rather quickly quenched by substrate excess, less abundant transcripts can often not be efficiently visualized even when applying TSA. The use of AP-Fast Blue fluorescent detection may provide a helpful alternative for fluorescent transcript visualization, as the AP reaction can proceed for extended times with a high signal-to-noise ratio. Our protocol thus provides a novel alternative for comparison of two different gene expression patterns in the embryonic zebrafish brain at a cellular level. The principles of our method were developed for use in zebrafish but may be easily included in whole-mount FISH protocols of other model organisms.