Labeling and imaging cells in the zebrafish hindbrain.
Labeling and imaging cells in the zebrafish hindbrain.
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DOI：
10.3791/1976
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发表时间：
2010-07-25
期刊：
影响因子：
--
通讯作者：
Brewster, Rachel
中科院分区：
文献类型：
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作者：
Jayachandran, Pradeepa;Hong, Elim;Brewster, Rachel
理解形成早期脊椎动物胚胎的形态发生过程的关键是高分辨率成像细胞的能力。在斑马鱼胚胎中，注射质粒DNA会导致镶嵌表达，从而可以看到单个细胞或小簇细胞(1)。我们描述了如何在普遍存在的启动子的控制下注射编码膜靶向绿色荧光蛋白(MGFP)的质粒DNA用于对正在进行神经分化的细胞进行成像。该协议的核心是以高分辨率对标记细胞进行切片和实时成像的方法。该方案需要将mGFP DNA注入斑马鱼幼崽的胚胎中。然后对胚胎进行振动切片、抗体标记和共聚焦显微镜成像。或者，表达mGFP的活胚胎可以使用延时共聚焦显微镜进行成像。我们之前已经使用这种简单的方法分析了驱动斑马鱼胚胎后脑区域神经管形成的细胞行为(2)。固定的准备使神经管中的细胞形状和组织得到了前所未有的可视化，而实时成像补充了这一方法，使人们能够更好地了解神经形成过程中发生的细胞动力学。
Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells (1) . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos (2). The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.