Name: imaging glycans in zebrafish embryos by metabolic labeling and bioorthogonal click chemistry
Uploaded: 2011-06-06T15:00:00
Description: Watch this Scientific Journal Video about Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry at JoVE.com

这项工作是部分支持由美国国立卫生研究院（GM093282到十五分; 3U54AI057158 - 06S1到RDS）和艾伯特爱因斯坦医学院的启动资金。

1。蛋的收集和Dechorionation <ol><li>收集和斑马鱼的卵转移到35mm培养皿中，删除尽可能多的水，然后添加1毫克/毫升Pronease在E3胚胎培养基E（5 mM氯化钠，0.17 mM的氯化钾，0.33毫米氯化钙2 · 2H 2 O，0.33毫米硫酸镁4，PH = 7.4），绒毛膜消化。 </li><li> 3-5分钟后，合并到一个烧杯中弥漫着鱼水（60毫克的“即时海洋”每公升蒸馏水H 2 O）的菜，轻轻的鸡蛋转移到烧杯中，并允...

<ol>
	<li>Laughlin, S. T., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+glycome.">Imaging the glycome.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 12-17 (2009).</li><li>Baskin, J. M., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioorthogonal+click+chemistry:+Covalent+labeling+in+living+systems.">Bioorthogonal click chemistry: Covalent labeling in living systems.</a> Qsar Comb. Sci. 26, 1211-1219 (2007).</li><li>Soriano del Amo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatible+copper(I)+catalysts+for+in+vivo+imaging+of+glycans.">Biocompatible copper(I) catalysts for in vivo imaging of glycans.</a> J. Am. Chem. Soc. 132, 16893-16899 (2010).</li><li>Chang, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+labeling+of+sialic+acids+in+living+animals+with+alkynyl+sugars.">Metabolic labeling of sialic acids in living animals with alkynyl sugars.</a> Angew. Chem. Int. Ed. 48, 4030-4033 (2009).</li><li>Laughlin, S. T., Baskin, J. M., Amacher, S. L., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+membrane-associated+glycans+in+developing+zebrafish.">In vivo imaging of membrane-associated glycans in developing zebrafish.</a> Science. 320, 664-667 (2008).</li><li>Baskin, J. M., Dehnert, K. W., Laughlin, S. T., Amacher, S. L., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+enveloping+layer+glycans+during+zebrafish+early+embryogenesis.">Visualizing enveloping layer glycans during zebrafish early embryogenesis.</a> Proc. Natl. Acad. Sci. U. S. A. 107, 10360-10365 (2010).</li><li>Kemp, H. A., Carmany-Rampey, A., Moens, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+chimeric+zebrafish+embryos+by+transplantation.">Generating chimeric zebrafish embryos by transplantation.</a> J. Vis. Exp. , (2009).</li><li>Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoenzymatic+synthesis+of+GDP-L-fucose+and+the+Lewis+X+glycan+derivatives.">Chemoenzymatic synthesis of GDP-L-fucose and the Lewis X glycan derivatives.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 16096-16101 (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <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+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J. Vis. Exp. , (2009).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> THE ZEBRAFISH BOOK, A guide for the laboratory use of zebrafish (Danio rerio). , (2007).</li><li>Kimmel, C. B., Warga, R. M., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+organization+of+the+zebrafish+fate+map.">Origin and organization of the zebrafish fate map.</a> Development. 108, 581-594 (1990).</li></ol>

<p class="jove_content"&gt;成像生物分子<em&gt;在体内</em提供在其原生环境的生物活动的重要见解。在这段视频中，我们将演示如何BTTES介导的生物相容性CuAAC通过显微注射与国内生产总值的FucAl一细胞阶段的胚胎和一个第二步荧光基团的共轭实现fucosylated聚糖在斑马鱼胚胎的包围层的标签<sup&gt; 3</sup&gt;。强大的标签可在2-3分钟内取得，并标记聚糖2.5 HPF早期检测。重要的是，国内生产总值的FucAl单剂量可以产生可检测的荧?...

<table border="1"><tbody><tr><td>试剂名称</td><td>公司</td><td>目录编号</td></tr><tr><td>铜（II）硫酸硫酸钠</td><td> Sigma - Aldrich公司</td><td> 203165 </td></tr><tr><td>的Alexa Fluor 488叠氮</td><td> Invitrogen公司</td><td> A10266 </td></tr><tr><td>右旋糖酐的Alexa Fluor 594 </td><td> Invitrogen公司</td><td> D - 22913 </td></tr><tr><td> （+） - L -抗坏血酸钠</td><td> Sigma - Aldrich公司</td><td> A7631 </td></tr><tr><td> Bathocuproinedisulfonic酸</td><td> Acros Organics公司</td><td> 164060010 </td></tr><tr><td>玻璃底部的微孔盘</td><td> MatTek </td><td> P35G - 1.5 - 14 - C </td></tr></tbody></table>

imaging glycans in zebrafish embryos by metabolic labeling and bioorthogonal click chemistry

活体成像聚糖最近已经启用了一个bioorthogonal化学记者战略治疗叠氮或炔标记的单糖1，2细胞或生物体。修改后的单糖，聚糖的生物合成机制处理，纳入细胞表面的糖复合物。 bioorthogonal叠氮或炔标签用于可视化的荧光探针，然后让共价键共轭，或用浓缩和glycoproteomic分析的亲和力探头。这个协议描述fucosylated聚糖在斑马鱼胚胎的无创成像通常使用的，包括程序：1）单细胞阶段的胚胎显微注射与国内生产总值5 alkynylfucose（国内生产总值FucAl），2）标签包围层fucosylated聚糖通过生物 ​​相容性铜（I）催化叠氮炔环加成反应（CuAAC），并通过共聚焦显微镜3 3）成像叠氮共轭荧光团的斑马鱼胚胎。这里描述的方法可以很容易地扩展到其他类别的聚糖，如含有唾液酸4 和 N -乙酰5，6的糖链，在发展中国家斑马鱼和其他生物可视化。

Watch this Scientific Journal Video about Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry at JoVE.com

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

点击化学的方法，允许炔标签聚糖在斑马鱼胚胎的快速，非侵入性和强大的标签描述。在这项研究中，已故的原肠胚形成阶段，在斑马鱼胚胎的包围层Fucosylated聚糖成像。

在斑马鱼胚胎的代谢标记和Bioorthogonal点击化学成像聚糖

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Research

JoVE Journal

Biology

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

科研

生物学

Generating Chimeric Zebrafish Embryos by Transplantation

移植嵌合斑马鱼胚胎生成

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

移植绿色荧光蛋白表达对视网膜和大脑发育的实时成像嵌合斑马鱼胚胎的卵裂球

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Imaging Cell Shape Change in Living Drosophila Embryos

Imaging Cell Shape Change in Living Drosophila Embryos

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

代谢途径的确认和发现通过 13 C - Proteinogenic氨基酸的标签

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

代谢标记和流式细胞仪测定细胞周期进程的动力学

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating ...

Metabolic Profile Analysis of Zebrafish Embryos

斑马鱼胚胎的代谢谱分析

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

富亮氨酸的代谢标记重复激酶1和2与放射性磷酸盐

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

Analysis of Oxidative Stress in Zebrafish Embryos

在斑马鱼胚胎氧化应激分析

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

组织特异性荧光的斑马鱼胚胎半自动成像

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...