Name: live-cell imaging and quantitative analysis of embryonic epithelial cells in xenopus laevis
Uploaded: 2010-05-23T17:00:00
Description: Watch this Scientific Journal Video about Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis at JoVE.com

这项工作是支持由一个国家卫生研究院（R01 HD044750），一个由美国国家科学基金会的事业补助金，并在美国心脏协会（格兰特在急救开始）的支持。作者感谢戴维森实验室成员：HY金，冯达索和J.周（负责日常实验室的帮助），和张琳为她在分子生物学和合成的mRNA的援助。我们承认，从本协议的某些部分，通过多年来的开发和测试的各种方法，作出了贡献的所有青蛙实验室（尤其是雷凯勒和Doug德西蒙的）试点工作。我们感谢理查德Harland和约翰沃林福德MEM - GFP和moesin - GFP质粒分别他们的实物礼品。

开始之前<ol class="lalpha"><li>皑皑的mRNA合成荧光标记的蛋白质和净化的mRNA（AmpliCap转录试剂盒;震中的生物技术，麦迪逊无线网络）的标准方法进行编码，从线性DNA模板。表达应为单一的使用时间在0.2 mL离心管分装，储存在-80 ° C </li><li>标准的方法来获得试管受精鸡蛋，，并去除鸡蛋皮已被描述以前 2 。前面3，一个程序，以获得从非洲爪蟾雌蛙卵已被证明。 在体外...

<ol>
	<li>Ninomiya, H., Winklbauer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+coating+controls+mesenchymal+shape+change+through+tissue-positioning+effects+and+reduction+of+surface-minimizing+tension.">Epithelial coating controls mesenchymal shape change through tissue-positioning effects and reduction of surface-minimizing tension.</a> Nat Cell Biol. 10 (1), 61-61 (2008).</li><li>Kay, B. K., Peng, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Xenopus laevis: Practical Uses in Cell and Molecular Biology. , (1991).</li><li>Cross, M. K., Powers, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obtaining+eggs+from+Xenopus+laevis+females.">Obtaining eggs from Xenopus laevis females.</a> J Vis Exp. , (2008).</li><li>Cohen, S., Au, S., Pante, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Xenopus+laevis+oocytes.">Microinjection of Xenopus laevis oocytes.</a> J Vis Exp. , (2009).</li><li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal Tables of Xenopus laevis (Daudin). , (1967).</li><li>Davidson, L. A., Ezin, A. M., Keller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+wound+healing+by+apical+contraction+and+ingression+in+Xenopus+laevis.">Embryonic wound healing by apical contraction and ingression in Xenopus laevis.</a> Cell Motil Cytoskeleton. 53 (3), 163-163 (2002).</li><li>Joshi, S. D., Dassow, M. v. o. n., Davidson, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+control+of+excitable+embryonic+tissues:+three+stimuli+induce+rapid+epithelial+contraction.">Experimental control of excitable embryonic tissues: three stimuli induce rapid epithelial contraction.</a> Exp Cell Res. 316 (1), 103-103 (2010).</li></ol>

我们已经介绍了实验方案，在我们的实验室中经常使用图像动态变化的根尖细胞皮层和摆动爪蟾胚胎中的上皮细胞的细胞膜骨架。应该使用这些协议作为出发点，为上皮细胞形状的生活成像;本议定书的优化是必不可少的，一些设置将改变视显微镜系统类型和类型的蛋白质之一，是在成像感兴趣。总之，该协议的步骤（植体隔离，成像和可视化的细胞和亚细胞成分，和定量方法）提供了一个...

live-cell imaging and quantitative analysis of embryonic epithelial cells in xenopus laevis

胚胎上皮细胞作为一个理想的模型来研究多细胞组织进行，如在细胞表面的面积和单元格的高度的变化，其几何形状的变化的形态发生，细胞进行有丝分裂和迁移。此外，上皮细胞也可以调节在癌旁组织的形态<sup&gt; 1</sup&gt;。传统的方法来研究上皮细胞和组织，涉及化学固定和组织学的方法来确定细胞形态或感兴趣的特定蛋白质的本地化。这些方法仍然是有用的细胞形态和组织架构，并提供“快照”，但是，仍然有许多了解细胞是如何获得特定形状，各种蛋白质如何移动或本地化具体岗位，和细胞遵循什么路径走向其最终差异化的命运。高分辨率实时成像传统方法的补充，也可以更直接进入调查涉及的形成，维护和形态发生的多细胞的上皮表的动态细胞过程。在这里，我们展示了从动物帽组织隔离的实验方法<em&gt;非洲爪蟾</em&gt;共焦成像的上皮细胞和简单的测量方法，可以增加上皮细胞的形态发生的分子和细胞研究的胚胎。

Watch this Scientific Journal Video about Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis at JoVE.com

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

非洲爪蟾胚胎上皮细胞是一个理想的模型系统来研究细胞极性的发展，并在上皮细胞形态发生改变形状等行为。传统的固定标本组织学正越来越多地辅以活细胞共聚焦成像。在这里，我们展示了隔离青蛙组织和可视化生活的上皮细胞和其骨架采用活细胞共聚焦显微镜的方法。

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Research

JoVE Journal

Biology

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Plastic Embedding and Sectioning of Xenopus laevis Embryos

塑料嵌入和非洲爪蟾胚胎切片

科研

生物学

Obtaining Eggs from Xenopus laevis Females

从非洲爪蟾女性获得卵子

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

制备及分馏爪蟾卵提取

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Microinjection of Xenopus Laevis Oocytes

非洲爪蟾卵母细胞的微量注射

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

组织测定动物第移植（ACT）的含量非洲爪蟾</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

在毛发再生的上皮 - 间充质信号的快速遗传分析

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Induction and Analysis of Epithelial to Mesenchymal Transition

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Facial Transplants in Xenopus laevis Embryos

Facial Transplants in Xenopus laevis Embryos

在面部移植<em&gt;非洲爪蟾</em&gt;胚胎

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

分析单个人类胚胎干细胞通过定量RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...