Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization
Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization
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DOI：
10.3791/51604
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发表时间：
2014-07-01
影响因子：
1.2
通讯作者：
Wingert, Rebecca A.
中科院分区：
文献类型：
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作者：
Cheng, Christina N.;Li, Yue;Wingert, Rebecca A.
斑马鱼胚胎现在通常用于基础和生物医学研究，以调查发育过程的遗传控制和模拟先天性异常。在生命的第一天，斑马鱼胚胎经历了许多发育阶段，包括受精、卵裂、原肠胚形成、分裂和器官发生，如肾脏、心脏和中枢神经系统。年轻斑马鱼胚胎的解剖结构为可视化和分析这些事件中涉及的组织提出了一些挑战，因为胚胎的发育与一个圆形的卵黄团有关。因此，为了准确分析和成像在尾芽和20体期（分别为受精后10和19小时（hpf））之间的固定胚胎标本的实验表型，例如使用全座原位杂交（WISH）染色的胚胎标本，通常需要将胚胎从卵黄球中取出并将其平放在玻璃载玻片上。但是，执行平面安装过程可能会很繁琐。因此，通过解剖技术的可视化演示，以及使用有助于最佳组织处理的试剂，极大地促进了成功和有效的平板安装制备。在这里，我们提供了我们的WISH方案，用于斑马鱼胚胎中基因表达的单色或双色检测，并演示了如何在这个染色固定标本的例子上进行平装程序。这种平装方案广泛适用于斑马鱼早期发育过程中出现的许多胚胎结构的研究，并且可以与固定胚胎样品上的其他染色方法一起实施。
The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.