我们想感谢在笼中的荧光素丹卡林帮助。此外，我们想感谢以下资金来源:范德比尔特大学医学发育生物学培训资助计划学校，江淮，和美国国立卫生研究院HD054534to JTG。

1。笼中的荧光素葡聚糖的合成<ol><li>测量出3.5 - aminodextran 4毫克，并添加到含1毫克的CMNB笼荧光SE的Invitrogen公司提供的有色管。在我们的手中，这一比例给出了一个〜2.5％，染料分子右旋糖酐的平均装载。 </li><li>加入500μL0.1 M的NA 2 B 4 O 7（四硼酸钠）缓冲区管。 </li><li>第旋涡30秒解散aminodextran笼荧光。 </li><li>让我们在涡旋混合器反应过夜。 </li><li>拧断则巴自旋列底部封闭和放松的上限。标记一个点上用毡尖笔列。将列在15 mL锥形管。 </li><li>在1000 XG离心机面临的转子中心的点为2分钟。压实后，立即使用列。 </li><li>放置在一个新的15 mL锥形管压实列。 </li><li>池反应混合物压实列树脂床的中心转移到。更换盖。 </li><li>在1000 XG离心机面临的转子中心的点为2分钟。分拆后，洗脱液和列的顶部将大致颜色相同的黄色阴影。 </li><li>黄色洗脱液（〜350μL）转移到1.5 ml离心管。 </li><li>冻干以干燥在speedvac。盖用铝箔纸speedvac。 </li><li>重新暂停导致〜苍白黄泡在水中2-3毫克到终浓度为1％W / V </li><li>在铝箔盖管，储存在-20 ° C的这一解决方案。这是一个好主意，以等分葡聚糖的解决方案，以防止重复冻融。 </li></ol> 2。笼中的荧光素葡聚糖注射到斑马鱼胚胎<ol><li>在0.2M氯化钾稀释1％W / V股票笼荧光葡聚糖1:5或1:10。 </li><li>注入0.5 - 笼入蛋黄细胞阶段的胚胎使用高压注射器的荧光解决方案1.0nL。一旦注入，胚胎离合器必须保持在28.5 ° C至减少非特异性uncaging在黑暗中。 </li><li>手动移除胚胎chorions。 </li><li>麻醉在4％tricaine的胚胎。 </li><li>胚胎将被安装在0.8％琼脂糖35mmx10mm培养皿中，然后盖上蛋水。 </li><li>至少15分钟，在50 ° C的热块平衡融化的琼脂糖。 </li><li>使用的玻璃吸管，吸取少量的水的一个胚胎，并释放到琼脂糖注:这是很重要的冷却琼脂糖管你的手，约30秒前把该管的胚胎，以避免杀死胚胎。 </li><li>移液器从试管胚胎，约琼脂糖50μL和弹出到培养皿的中心内容。 </li><li>快速定位与uncaged面临细胞的胚胎，使用一个塑料螺纹注 :酷很快，所以仓促定向胚胎琼脂糖。如果操纵琼脂糖变得僵硬后，可能会损坏胚胎。 </li><li>允许琼脂糖完全巩固（这需要几分钟），并填写菜三分之二与鸡蛋的水含有tricaine 4％和0.003％机动部队。 </li></ol> 3。激光荧​​光素葡聚糖Uncaging <ol><li> uncaging使用显微镜必须有一个40倍的水浸泡的目标。 </li><li>我们用365 nm的染料细胞和70％/ 30％的分裂分色镜的光电子仪器的激光。 </li><li> uncaging之前，激光必须作出的目标和衰减到合适的电源的齐焦。 </li><li>要集中的激光，将根据客观镜像玻片上，重点目标，直到在幻灯片的划痕可见。 </li><li>调整激光衰减器，直到激光可以产生清晰可见，在单脉冲镜从头。 </li><li>向上和向下调整的目标重点。如果激光可以产生了一个从无到有的目标是重点，调整激光的聚焦¼交替上升或下降。重复，直到激光可以从头开始的目标是只有当聚焦镜。 </li><li>根据uncaged面积的目标和重点地点安装胚胎。 </li><li>要uncage，着眼于利益的一个单元格，并在2-3赫兹脉冲的10-20倍。 </li><li> uncaging后，免费的胚胎剥离琼脂糖钳和把鸡蛋水含有0.003％机动部队。让胚胎的发展，直到它们被固定在光紧框。 </li></ol> 4。抗体标记的Uncaged荧光素葡聚糖检测<ol><li>修复胚胎在AB固定液（4％多聚甲醛，氯化钙为0.3mm 2，8％的蔗糖，1X的磷酸盐缓冲生理盐水，pH值7.3），两到四个小时，在室温（RT）或隔夜（O / N）在4 ° C所有标签的​​步骤，应保持轻紧框或铝箔包裹管的胚胎。 </li><li>删除固定液，用100％甲醇取代，在室温下10分钟离开，除去甲醇和replaCE新鲜100％甲醇。 </li><li>商店在-20 ° C，在甲醇至少30 分钟注 :您可以存储长达两个星期之前抗原表位在甲醇开始降低。 </li><li>补充水分胚胎在室温洗涤5分钟，75％MeOH/1X磷酸盐缓冲生理盐水，0.1％吐温20（1X PBSTw），然后50％MeOH/50％1XPBTw，然后MeOH/75％1X PBSTw 25％。 </li><li>在1XPBSTw 5分钟内清洗三次。 </li><li>如果胚胎&gt; 24小时，permiabilize 10μg/ mL的蛋白酶K在1XPBSTw胚胎。 Permiabilization时间是在舞台上的依赖。五分钟蛋白酶K是足够的，如果胚胎受精后（HPF）是24至48小时。如果年纪较大的幼虫，可能需要更多的时间。重新修复抗体固定液在室温下20分钟</li><li>在1XPBSTw 5分钟，在室温下清洗5次。 </li><li>孵育块解决方案（1XPBS，0.1％的Triton X100，10％羊血清，10％的牛血清蛋白，1％二甲基亚砜）在室温下1-2小时。 </li><li>封闭液稀释的抗体（抗荧光素加抗细胞类型特异性标志物抗体）。 </li><li>孵育胚胎O / N在4 ° C在含初级抗体的解决方案。 </li><li>清洗四次，在1XPhosphate 20分钟的缓冲生理盐水，0.1％的Triton X100（PBSTx）在室温下。 </li><li>在封闭液稀释的荧光标记抗体（通常为1:300）。 </li><li>孵育O / N在4 ° C在含有二级抗体的解决方案。 </li><li>在室温下在1XPBSTx 20分钟清洗四次。 </li><li>清除在50％glycerol/50％，在室温下至少有两个小时1XPBS的胚胎，然后取出甘油/ PBS混合，添加100％的甘油，让站在O / N 4 ° C。 </li></ol> 5。代表性的成果: 免疫标记后，应该有一个uncaged（Figure.1）细胞染色丰富的区域。这是很平常看到一个比较高的信噪比&gt; 48小时老斑马鱼，由于荧光素的自发uncaging。 <img src="/files/ftp_upload/2672/2672fig1.jpg" alt="图1" /> 图1。传承的斑马鱼松果体复杂的标签。 HPF，24岁的前松果体原基的部分，foxd3表示:GFP的表达，uncaged使用紫外激光脉冲通过48hpf），左路集群的细胞称为parapineal（红色圆圈）已经摆脱了uncaged域（白色圆圈），B）Uncaged的荧光信号是丰富parapineal（红圈）和松果体器官（白色圆圈） 。

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	<li>Hatada, Y., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fate+map+of+the+epiblast+of+the+early+chick+embryo.">A fate map of the epiblast of the early chick embryo.</a> Development. 120, 2879-2889 (1994).</li><li>Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+of+the+blastomeres+of+the+32-cell-stage+Xenopus+embryo.">Fates of the blastomeres of the 32-cell-stage Xenopus embryo.</a> Dev Biol. 122, 300-319 (1987).</li><li>Wetts, R., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+Precursors+Can+Give+Rise+to+All+Major+Cell-Types+of+the+Frog+Retina.">Multipotent Precursors Can Give Rise to All Major Cell-Types of the Frog Retina.</a> Science. 239, 1142-1145 (1988).</li><li>Wetts, R., Fraser, S. E. <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+fluorescent+tracers+to+study+neural+cell+lineages.">Microinjection of fluorescent tracers to study neural cell lineages.</a> Development. , 1-8 (1991).</li><li>Woo, K., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Order+and+coherence+in+the+fate+map+of+the+zebrafish+nervous+system.">Order and coherence in the fate map of the zebrafish nervous system.</a> Development. 121, 2595-2609 (1995).</li><li>Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proc Natl Acad Sci U S A. 99, 12651-12656 (2002).</li><li>Sato, T., Takahoko, M., Okamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HuC:Kaede,+a+useful+tool+to+label+neural+morphologies+in+networks+in+vivo.">HuC:Kaede, a useful tool to label neural morphologies in networks in vivo.</a> Genesis. 44, 136-142 (2006).</li><li>Wiedenmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EosFP,+a+fluorescent+marker+protein+with+UV-inducible+green-to-red+fluorescence+conversion.">EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion.</a> Proc Natl Acad Sci U S A. 101, 15905-15910 (2004).</li><li>Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Fluorescent Probes and Research Chemicals. , 707-711 (2002).</li><li>Kozlowski, D. J., Murakami, T., Ho, R. K., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+cell+movement+and+tissue+patterning+in+the+zebrafish+embryo+revealed+by+fate+mapping+with+caged+fluorescein.">Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein.</a> Biochem Cell Biol. 75, 551-562 (1997).</li><li>Kozlowski, D. J., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivatable+(caged)+fluorescein+as+a+cell+tracer+for+fate+mapping+in+the+zebrafish+embryo.">Photoactivatable (caged) fluorescein as a cell tracer for fate mapping in the zebrafish embryo.</a> Methods Mol Biol. 135, 349-355 (2000).</li><li>Russek-Blum, N., Nabel-Rosen, H., Levkowitz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+fate+map+of+the+zebrafish+diencephalon.">High resolution fate map of the zebrafish diencephalon.</a> Dev Dyn. 238, 1827-1835 (2009).</li><li>Summers, R. G., Piston, D. W., Harris, K. M., Morrill, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+orientation+of+first+cleavage+in+the+sea+urchin+embryo,+Lytechinus+variegatus,+does+not+specify+the+axes+of+bilateral+symmetry.">The orientation of first cleavage in the sea urchin embryo, Lytechinus variegatus, does not specify the axes of bilateral symmetry.</a> Dev Biol. 175, 177-183 (1996).</li></ol>

如上所述，这个协议提供了相对快速的血统在斑马鱼标记法，显微注射法，显微镜，免疫后的常用技巧。我们发现，激光uncaging是最有效和最具成本效益的方式uncage在本地化时尚的荧光。这种方法可用于实验端点4 DPF后期谱系标签。然而，细胞分裂时，每个单元的笼荧光素浓度，最终跌幅超过检测水平。此外，已经出现了自发的，非特异性荧光uncaging的斑马鱼幼虫 11的报告。因此，u​​ncag...

A correction was made to <a href="http://jove.unicartagenaproxy.elogim.com/details.php?id=2672">Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran</a>. There was an error in the authors, Ilya A. Shestopalov and James K. Chen, names. The author's names have been corrected to: Ilya A. Shestopalov and James K. Chen instead of: Ilya Shestopalov and James Chen

A correction to the article Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran was made.

Erratum: Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

<table><tbody><tr><td>CMNB-caged fluorescein SE</td><td>Invitrogen</td><td>C20050</td><td></td></tr><tr><td>Aminodextran, 10kDa</td><td>Invitrogen</td><td>D1860</td><td></td></tr><tr><td>Zeba desalt spin columns</td><td>Pierce, Thermo Scientific</td><td>89889</td><td></td></tr><tr><td>goat anti-fluorescein antibody</td><td>Invitrogen</td><td>A11095</td><td></td></tr><tr><td>Alexa Fluor 633 Donkey anti-goat antibody</td><td>Invitrogen</td><td>A21082</td><td></td></tr><tr><td>35mmx10mm petri dishes</td><td>BD Biosciences</td><td>351008</td><td></td></tr><tr><td>Upright compound microscope with 40x water immersion objective</td><td>Leica Microsystems</td><td>DM6000B</td><td></td></tr><tr><td>MicroPoint Manual Laser Illumination System</td><td>Photonic Instruments</td><td></td><td></td></tr><tr><td>Phenylthiourea (PTU)</td><td>Alfa Aesar</td><td>L06690</td><td></td></tr><tr><td>Tricaine</td><td>Sigma-Aldrich</td><td>E10521</td><td></td></tr><tr><td>Proteinase K</td><td>Roche Group</td><td>03115836991</td><td></td></tr><tr><td>Plastic thread</td><td>Any Supplier</td><td></td><td></td></tr><tr><td>Agarose</td><td>Research Products International Corp.</td><td>A20090</td><td></td></tr><tr><td>SpeedVac</td><td>Thermo Fisher Scientific, Inc.</td><td>Savant SPD131DDA</td><td></td></tr><tr><td>Vortexing mixer</td><td>VWR international</td><td>Vortex Genie 2</td><td></td></tr><tr><td>Pressure injector</td><td>Applied Scientific Instrumentation</td><td>MPPI-3</td><td></td></tr></tbody></table>

lineage labeling of zebrafish cells with laser uncagable fluorescein dextran

发育生物学中的一个核心问题是演绎出无数的细胞类型，目前他们未分化的前体出现在脊椎动物的起源。研究人员所采用的谱系标签的各种方法，如DII 标签 1和压力注射溯源酶 2的模型系统开发的后期阶段，以确定细胞的命运。在斑马鱼（ 斑马鱼 ）的第一命运地图iontophoretic注射荧光染料如罗丹明右旋糖酐，组装，成单细胞在胚胎的离散地区和追踪标记细胞的命运，随着时间的推移3-5。这些方法虽然有效，技术要求高，需要专门的设备，不常用的斑马鱼实验室发现。最近，photoconvertable荧光蛋白，如EOS和枫，不可逆转的交换机从绿色变为红色荧光，当暴露在紫外线，增加使用在斑马鱼 6-8 。光学清晰度的转基因斑马鱼的胚胎和相对缓和这些谱系标签特别有吸引力的工具，并观察细胞的迁移，在体内7。尽管它们的效用，这些蛋白质有一些缺点相比，染料介导的宗族标记方法。最关键的是我们在发现这些蛋白质的光转化过程中获得3 - D分辨率高的困难。在这一点上，也许在斑马鱼的血统标签的决议和易用性的最佳结合 ​​，使得使用笼荧光葡聚糖，一种荧光染料，势必淬火组口罩荧光9。染料就可以"uncaged"（从淬火组发布）在一个特定的细胞使用激光或水银灯紫外线光，使荧光或免疫检测的可视化。笼荧光iontophoretic方法不同，可注射用标准的注射器具，并配备了一个针孔10 epifluorescence显微镜uncaged。此外，对荧光抗体检测只有uncaged形式，和抗原表位生存固定以及11。最后，笼中的荧光可以被激活，具有非常高的3 - D分辨率，特别是如果采用双光子显微镜12,13。这个协议描述笼荧光和激光uncaging宗族标签的方法。 Subsequenctly，uncaged荧光检测如GFP的其他抗原表位，同时通过与抗体标签。

Watch this Scientific Journal Video about Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran at JoVE.com

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

该协议描绘的方式，标签和跟踪的小斑马鱼的胚胎细胞群体，使用紫外线uncaging笼荧光，整个装载免疫标记uncaged荧光信号放大的命运。

天堂斑马鱼的细胞标记与激光Uncagable荧光葡聚糖

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

lineage-labeling-zebrafish-cells-with-laser-uncagable-fluorescein

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.8K 次观看.  Vanderbilt University. 该协议描绘的方式，标签和跟踪的小斑马鱼的胚胎细胞群体，使用紫外线uncaging笼荧光，整个装载免疫标记uncaged荧光信号放大的命运。

视频: Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

Labeling and Imaging Cells in the Zebrafish Hindbrain

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.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

在斑马鱼后脑细胞标记和成像

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

科研

神经科学

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

发育生物学

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

<meta charset="utf8"></head><body>使用 CRISPR/Cas9 敲入在斑马鱼幼虫中进行荧光蛋白标记

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in vivo imaging due to its transparency during the early developmental stage. Here, we provide a detailed protocol for performing high-efficiency fluorescence gene KI, rapid screening for KI founders, and low-abundance protein tracing in zebrafish larvae, which will lay a critical foundation for subsequent physio-pathological studies in zebrafish. The current protocol includes complete steps for the sgRNA design for the gene of interest, sgRNA in vitro transcription, Cas9 mRNA in vitro transcription, in vivo sgRNA screen for the one with the highest efficiency, donor plasmid design and construction, microinjection in zebrafish larvae, KI founder screen and zebrafish live imaging. Critical steps, troubleshooting tips, quality control methods, and advantages and applications of this protocol are included and discussed. This protocol assures quick and accurate results at a low cost and has been validated by multiple trials.

<meta charset="utf8"></head><body>荧光标记以可视化斑马鱼中的低表达蛋白质

Here, we present a protocol to label proteins with a fluorescence protein tag in zebrafish larvae, a newly developed and modified in vivo system especially useful for visualizing low-abundance proteins in zebrafish.

荧光标记以可视化斑马鱼中的低表达蛋白质

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in ...

遗传学

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

在斑马鱼胚胎的单个神经元和神经嵴细胞的细胞运动和肌动蛋白细胞骨架的实时成像

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

生物学

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

天堂斑马鱼的细胞标记与激光Uncagable荧光葡聚糖

摘要

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此视频中的章节

在斑马鱼后脑细胞标记和成像

在活斑马鱼胚胎成像的亚细胞结构

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

荧光标记以可视化斑马鱼中的低表达蛋白质

荧光标记以可视化斑马鱼中的低表达蛋白质

荧光标记以可视化斑马鱼中的低表达蛋白质

在斑马鱼胚胎的单个神经元和神经嵴细胞的细胞运动和肌动蛋白细胞骨架的实时成像

在现场的斑马鱼胚胎的基于双光子Photoactivation

天堂斑马鱼的细胞标记与激光Uncagable荧光葡聚糖

摘要

探索更多视频

此视频中的章节

在斑马鱼后脑细胞标记和成像

在活斑马鱼胚胎成像的亚细胞结构

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

荧光标记以可视化斑马鱼中的低表达蛋白质

荧光标记以可视化斑马鱼中的低表达蛋白质

荧光标记以可视化斑马鱼中的低表达蛋白质

在斑马鱼胚胎的单个神经元和神经嵴细胞的细胞运动和肌动蛋白细胞骨架的实时成像

在现场的斑马鱼胚胎的基于双光子Photoactivati​​on

在现场的斑马鱼胚胎的基于双光子Photoactivation