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，并允许鸡蛋到入水“落“。 </li><li>鱼水三次冲洗的鸡蛋。从他们的绒毛膜大部分的鸡蛋会被释放。 </li><li>使用火抛光的玻璃巴斯德吸管，转让dechorionated鸡蛋琼脂糖涂层的培养皿菜肴充满E3胚胎中期。 </li></ol> 2。显微注射与国内生产总值FucAl <ol><li>以下报道的协议 7，准备注射菜。 </li><li>转移到充满E3胚胎中期注射菜dechorionated鸡蛋。 </li><li>准备针，2μL注射液和负载。该解决方案包含了20毫米的国内生产总值FucAl合成chemoenzymatically 8任的Alexa Fluor 594 -葡聚糖（5％W / V）作为示踪或酚红样染料（0.1％W / V）在0.2 mol氯化钾。作为阴性对照，与GDP -岩藻糖注射液取代国内生产总值FucAl。 </li><li>打破了针，调整喷油压力和持续时间，产生了1 NL下降9。 </li><li>注入1 NL的任何一个解决方案的鸡蛋。 </li><li>转移到琼脂糖涂层培养皿充满E3胚胎中期的菜蛋。 </li><li>在28 ° C孵育的鸡蛋和未受精的卵受精后三到四个小时内删除。 </li></ol> 3。 BTTES - 铜（I）催化点击化学反应<ol><li>当胚胎达到理想的发育阶段（如已故的原肠胚，组织分割和早期幼虫），外套一个96孔板，用琼脂糖基地。 </li><li> ，每孔加入92μLE3胚胎中期，随后另外4μL的Alexa Fluor 488叠氮（从一个2.5毫米的股票，在H 2 O），2μLBTTES，硫酸铜4 6:1复杂，和轻轻摇动混合。 </li><li>点击化学试剂使用火抛光的玻璃巴斯德吸管转移到胚胎。每口井应包含少于五个胚胎。 </li><li>添加2.5μL新鲜配制的抗坏血酸钠（100毫米股票在H 2 O）发起的点击反应 3 。终浓度为每一个试剂的Alexa Fluor 488叠氮：100微米; 硫酸铜 4：50微米; BTTES：300微米;抗坏血酸钠：2.5毫米。 </li><li> 3分钟后，加入2微升bathocuproine磺酸（H 2 O 50毫米股票），生物相容性铜螯合剂，然后立即稀释100μLE3胚胎中期淬火反应。 </li><li>胚胎玻璃培养皿和清洗处理的胚胎与15毫升的E3胚胎中期的2倍。 </li></ol> 4。成像<ol><li> MatTek玻璃底部的微孔盘上放置一个超低熔点琼脂糖（E3胚胎中期的1.2％（W ​​/ V））的下降，胚胎放入琼脂糖下降。 </li><li>位置的胚胎背侧或外侧，并置于冰上5分钟，以巩固琼脂糖下降的菜。 E3胚胎培养基添加轻轻的菜，直到它涵盖了琼脂糖下降。 </li><li>顺序使用共聚焦显微镜，荧光和明亮的现场图像采集。所有胚胎图像采集使用一个5微米的一步区间。综合数字准备使用ImageJ软件。 </li></ol> 5。代表性的成果图1显示了我们两个步骤的标签战略的工作流程。图2显示了斑马鱼的胚胎在受精后9.5小时（HPF）通过BTTES介导CuAAC标签。 BTTES是一个三（triazolylmethyl）胺为基础的配体。它加速CuAAC显着时在原位生成铜（一）协调，促进在生物系统中的环加成反应迅速没有明显的毒性。紧接着一个3分钟的点击反应，我们能够探测到强大的国内生产总值FucAl处理的胚胎标签（图2，左侧面板）。只有背景荧光检测控制胚胎显微注射与GDP -岩藻糖（图2中，右图）。 <img src="/files/ftp_upload/2686/2686fig1.jpg" alt="图1" /> 图1标签fucosylated聚糖在斑马鱼胚胎的包围层的战略。 <img src="/files/ftp_upload/2686/2686fig2.jpg" alt="图2" /> 图2。fucosylated聚糖通过BTTES铜（I）催化点击化学斑马鱼胚胎发育期间体内成像。单细胞阶段斑马鱼的胚胎显微注射单剂量的国内生产总值FucAl并允许发育运到9.5 HPF。胚胎的Alexa Fluor 488叠氮BTTES铜（一）催化反应。使用共聚焦显微镜成像反应胚胎。最大强度Z -投影图像的Alexa Fluor 488荧光（上图）;明场（下图）。比例尺：100微米。 故障排除<table border="1"><tbody><tr><td>问题</td><td>原因</td><td>补救</td></tr><tr><td>看不健康的胚胎</td><td>显微注射的溶液中含有污染物</td><td>核苷酸糖的纯度应大于85％。检查你的源代码。 </td></tr><tr><td>没有标签后的反应</td><td>铜浓度低于30微米</td><td>要小心，不要稀释反应液加​​入过量E3胚胎培养基加入胚胎时，反应率显着下降，当铜浓度低于30微米的。 </td></tr><tr><td>胚胎死亡后的反应</td><td>胚胎处理不当</td><td>确保吸管火抛光和反应容器涂一层非常薄的0.5％琼脂糖。 </td></tr><tr><td>图像看起来参差不齐</td><td>所用试剂不正确混合后再加入胚胎</td><td>按照建议的顺序添加试剂，并确保点击化学试剂已正确之前加入胚胎混合。 </td></tr><tr><td>图像显示，胚胎受损</td><td>剧烈摇晃的​​胚胎损害的反应过程中</td><td>一旦胚胎是在溶液中轻轻摇动少于10秒。 </td></tr><tr><td></td><td></td><td></td></tr></tbody></table>

<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 ...

imaging-glycans-zebrafish-embryos-metabolic-labeling-bioorthogonal

Research

JoVE Journal

Biology

13.5K 次观看.  Albert Einstein College of Medicine, Yeshiva University. 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。

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

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

科研

生物学

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

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

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.  ...

Generating Chimeric Zebrafish Embryos by Transplantation

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 organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

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

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

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

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

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 ...

Measuring Cell Cycle Progression Kinetics with Metabolic Labeling and Flow Cytometry

Precise control of the initiation and subsequent progression through the various phases of the cell cycle are of paramount importance in proliferating cells. Cell cycle division is an integral part of growth and reproduction and deregulation of key cell cycle components have been implicated in the precipitating events of carcinogenesis 1,2. Molecular agents in anti-cancer therapies frequently target biological pathways responsible for the regulation and coordination of cell cycle division 3. Although cell cycle kinetics tend to vary according to cell type, the distribution of cells amongst the four stages of the cell cycle is rather consistent within a particular cell line due to the consistent pattern of mitogen and growth factor expression. Genotoxic events and other cellular stressors can result in a temporary block of cell cycle progression, resulting in arrest or a temporary pause in a particular cell cycle phase to allow for instigation of the appropriate response mechanism. 
The ability to experimentally observe the behavior of a cell population with reference to their cell cycle progression stage is an important advance in cell biology. Common procedures such as mitotic shake off, differential centrifugation or flow cytometry-based sorting are used to isolate cells at specific stages of the cell cycle 4-6. These fractionated, cell cycle phase-enriched populations are then subjected to experimental treatments. Yield, purity and viability of the separated fractions can often be compromised using these physical separation methods. As well, the time lapse between separation of the cell populations and the start of experimental treatment, whereby the fractionated cells can progress from the selected cell cycle stage, can pose significant challenges in the successful implementation and interpretation of these experiments.
Other approaches to study cell cycle stages include the use of chemicals to synchronize cells. Treatment of cells with chemical inhibitors of key metabolic processes for each cell cycle stage are useful in blocking the progression of the cell cycle to the next stage. For example, the ribonucleotide reductase inhibitor hydroxyurea halts cells at the G1/S juncture by limiting the supply of deoxynucleotides, the building blocks of DNA. Other notable chemicals include treatment with aphidicolin, a polymerase alpha inhibitor for G1 arrest, treatment with colchicine and nocodazole, both of which interfere with mitotic spindle formation to halt cells in M phase and finally, treatment with the DNA chain terminator 5-fluorodeoxyridine to initiate S phase arrest 7-9. Treatment with these chemicals is an effective means of synchronizing an entire population of cells at a particular phase. With removal of the chemical, cells rejoin the cell cycle in unison. Treatment of the test agent following release from the cell cycle blocking chemical ensures that the drug response elicited is from a uniform, cell cycle stage-specific population. However, since many of the chemical synchronizers are known genotoxic compounds, teasing apart the participation of various response pathways (to the synchronizers vs. the test agents) is challenging. 
Here we describe a metabolic labeling method for following a subpopulation of actively cycling cells through their progression from the DNA replication phase, through to the division and separation of their daughter cells. Coupled with flow cytometry quantification, this protocol enables for measurement of kinetic progression of the cell cycle in the absence of either mechanically- or chemically- induced cellular stresses commonly associated with other cell cycle synchronization methodologies 10. In the following sections we will discuss the methodology, as well as some of its applications in biomedical research.

Tracking subtle changes in the progression and kinetics of cell cycle stages can be accomplished by use of a combination of metabolic labeling of nucleic acids with BrdU and total genomic DNA staining via Propidium Iodide. This method avoids the need of chemical synchronization of cycling cells, thereby preventing the introduction of non-specific DNA damage, which in turn affects cell cycle progression.

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

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

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 ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

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

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for 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 ...