这项工作是由美国国立卫生研究院授予授予R.布鲁斯特（1R01GM085290 - 01A1）支持。

1.Microinjection <ol><li>稀释质粒编码的膜有针对性的绿色荧光蛋白（mGFP，礼貌理查德哈兰德）至40毫微克/毫升的浓度。 DNA是由线性质粒（mGFP/PCS2，礼貌理查德哈兰德）纯化使用Qiagen公司马克西准备包准备。酚红（稀释后总体积的1 / 10）的解决方案是首选的可视化解决方案。 DNA溶液置于冰上。 </li><li>在立体显微镜下，校准的注射针（90毫米Narishinge科学的玻璃毛细管，猫编号NA - GD - 1），对准在毕业的幻灯片针，轻轻拍打针直径为1微米的部分，用一个干净的剃刀刀片（获得一个适当大小的一角）。 </li><li>为了校准针注入已知体积（2nl），显微注射器具的针连接（PCI 100微量;哈佛器械）和前填写含有酚红和水放在一块解决方案0.5μL针石蜡。该解决方案可免除使用脚踏板。设置至7千帕的压力和不同的时间（10毫秒30msec），因此，它需要250踏板的解决方案，以免除0.5μL。 </li><li>在加湿100毫米的培养皿中的蜡地带，小心地校准针。加湿的菜，将周围的边缘湿Kimwipes（这可防止蒸发解决针）。 </li><li>使用微针的针尾（未校准结束）加载到0.5 -加入1μl的DNA。 </li><li>一旦该解决方案已经达到了针头，连接加载到显微注射器具使用的针。微量调整设置，以提供约2 / NL注射用脚踏。 </li><li>下一步，位置在中间充满了胚胎中等（1.0毫升汉克的联合1＃，比较上升零点一毫升汉克的股票2＃，1.0毫升汉克的股票＃4，95.9毫升DDH 2 Ø，1.0毫升汉克的库存＃5一个基于Petri菜斑马鱼胚胎1.0毫升新鲜汉克的股票＃6，使用约10滴1 M NaOH溶液pH值7.2）。 </li><li>一旦胚胎发展所需的阶段，用细镊子轻轻夹住绒毛膜和位于进针注射针对性的区域。对于广泛mGFP的表达，注入1cell阶段的蛋黄或细胞质DNA。要限制mGFP表达到一个较小的区域，在4个（或更多）细胞阶段注入到一个单细胞的细胞质DNA。 </li><li>按显微注射器具的脚踏板，并提供约2 NL，确保该解决方案（可见由于酚红此外）是正确的注入。 </li><li>小心取出胚胎针。 </li><li>在培养皿中的所有胚胎重复。 </li><li>允许胚胎在28.5 ° C所需的阶段。 </li><li>轻轻剥落绒毛膜使用罚款钳（如果胚胎24hpf年轻，在玻璃培养皿dechorionating增加胚胎的成活率）Dechorionate胚胎。 </li><li>从培养皿菜使用的玻璃吸管取出空蛋壳。 </li></ol> 2。 vibratome成像mGFP标记的部分<ol><li>使用玻璃吸管运输到固定液（4％多聚甲醛在1X的磷酸盐缓冲液（PBS）的胚胎。固定在4 ° C过夜胚胎。 </li><li>胚胎3次在1X PBS洗，每次5分钟。 </li><li>放置在培养皿中，用细镊子胚胎，除去蛋黄，不破坏胚胎的情况下。将尽可能多蛋黄。 </li><li>准备在H 2 O 4％低熔点琼脂糖</li><li>塑料切片模具放入熔化的琼脂糖。 </li><li>用细镊子解除从培养皿中注入胚胎，并放置在琼脂糖东方。胚胎应定位于对模具的一端。 </li><li>琼脂糖硬化后，用一个平面工具，如压舌板，从模具取出琼脂糖块。 </li><li>琼脂糖块用刀片切底，以便它更是。 </li><li>添加超级胶水下降的vibratome阶段，和地方上的琼脂糖块（块被夷为平地剃刀方应面对胶水）。对于多个胚胎切片，安装在舞台上的几个琼脂糖包埋标本。 </li><li>将vibratome阶段的vibratome（Vibratome公司）。 </li><li>设置参数的vibratome常用的设置（速度3，厚度为50μm）。如果切片多个胚胎，在舞台上保持一致的琼脂糖块，并确保刀片穿过整个胚胎，但没有通过琼脂糖块的整个宽度（例如，对应相同的胚胎部分将留在一起）。 </li><li>倒入1X PBS vibratome阶段。 </li><li>科。 </li><li>一旦胚胎是sectio定义，删除琼脂糖块（S）从vibratome阶段。 </li><li>仔细分离切割琼脂糖块（S）的背面，每个部分。 </li><li>用细镊子选择所需的部分和地方填补一个PBS - 1X多井的菜（使用不同的部分对应不同的胚胎井）。 </li><li>重复每个胚胎。 </li><li>着手抗体标记，虽然23步骤19，加强mGFP成像（这一步可能没有必要的，如果成像在一天之内或准备两个部分）。 </li><li>在1X PBS阻塞的解决方案，以阻止非特异性结合，在室温下处理30分钟部分。 </li><li>一抗（兔A - GFP，Invitrogen公司的猫A11122号）：1:200稀释后在室温下为5小时或隔夜在振动筛上的一个封闭液在4 ° C孵育章节</li><li>在1％BSA/1X PBS / 1％DMSO溶液清洗4次（10分钟，15分钟，30分钟和60分钟，每个）。 </li><li>治疗与适当的封闭液中的二级抗体在室温下为5小时或隔夜节于4 ° C。 </li><li>在1％BSA/1X PBS / 1％DMSO溶液清洗4次（10分钟，15分钟，30分钟和60分钟，每个）。 </li><li>治疗用DAPI（0.1％的DAPI于50ml 1X PBS; Invitrogen公司CAT号D1306）的部分为30分钟细胞核染色。 </li><li>在1X PBS三次，每次5分钟洗净。 </li><li>安装上滑动。 </li><li>利用共聚焦显微镜图像。 </li></ol> 3。实时成像<ol><li> Dechorionate生活，注入胚胎前30分钟，他们达不到预期的发展阶段。镇静的解决方案中的胚胎（0.01％Tricaine在胚胎中期）放置15分钟。 </li><li>放置一个35mm的玻璃底培养皿下降了1％琼脂糖（MatTek;零件编号P35G - O - 20 - C）。 </li><li>允许琼脂糖巩固。 </li><li>热毛细管以上的火焰，使琼脂糖3孔（可用于其他对象，如微量提示，使孔）。确保在孔底，没有残留的琼脂糖。 </li><li>与胚胎中等填充的培养皿中。 </li><li>使用玻璃吸管放置在洞口的胚胎。 </li><li>接下来，东方的胚胎，用细镊子，使该地区进行成像（背头）与玻璃接触。 </li><li>仔细运输培养皿显微镜阶段（放置在培养皿中，以防止水分蒸发的盖子）。 </li><li>虽然成像，确保胚胎在28.5 ° C。 </li></ol> 4。结果我们在这里介绍一个简单的方法，图像的单个细胞在斑马鱼神经管使用马赛克表达。尽管斑马鱼胚胎的光学透明性，我们发现，神经细胞的可视化是在使用一个vibratome获得截面大大增强。这些部分厚（50毫米）允许从一个给定的细胞在不同的使用共聚焦显微镜焦平面成像蜂窝扩展。结果使用这种方法已在其他地方发表了 2，但代表的图像在一个WT胚胎（图1）和N -钙粘蛋白（N - CAD）的神经管细胞突变（图2）提供。后者揭示了该细胞中的突变体不能面向中线（图2）正常的神经板在N - CAD外侧地区。在这个镶嵌标签对比，与一般的细胞表面标志物，如β- catenin的神经上皮免疫不允许单个细胞形态的可视化（图3）。 时间失效成像活胚胎补充镶嵌固定标本的标签，从而使更好地理解了在神经胚细胞动力学。电影1显示，野生型细胞中线积极争取通过扩大内侧面向的膜突起迁移。 <img src="/files/ftp_upload/1976/1976fig1.jpg" alt="图1" /> 图1。mGFP表达一个WT斑马鱼胚胎的神经管。 <img src="/files/ftp_upload/1976/1976fig2.jpg" alt="图2" /> 图2。mGFP表达在一个N - cadherin的斑马鱼胚胎的神经管。 <img src="/files/ftp_upload/1976/1976fig3.jpg" alt="图3" /> 图3，B - catenin的免疫染色的神经上皮。

<ol>
	<li>N&#252;sslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish Practical approach series. , (2002).</li><li>Hong, E., Brewster, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-cadherin+is+required+for+the+polarized+cell+behaviors+that+drive+neurulation+in+the+zebrafish.">N-cadherin is required for the polarized cell behaviors that drive neurulation in the zebrafish.</a> Development. 133, 3895-3905 (2006).</li><li>Andersen, E., Asuri, N., Clay, M., Halloran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+Cell+Motility+and+Actin+Cytoskeleton+of+Individual+Neurons+and+Neural+Crest+Cells+in+Zebrafish+Embryos.">Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos.</a> J Vis Exp. , (2010).</li></ol>

总之，描述标签技术在这里允许在斑马鱼的胚胎形成过程的单细胞分析。此协议的主要重点是对成像标记在使用一个无处不在的启动子控制下的mGFP的神经管细胞的方法。对于这种瞬时表达检测读者的额外的应用程序应该是指一个由安德森等人最近的一篇文章 。3 。

A correction was made to <a href="http://jove.unicartagenaproxy.elogim.com/index/Details.stp?ID=1976">Labeling and Imaging Cells in the Zebrafish Hindbrain</a>. There was an error in the authors affiliations. The authors have been corrected to:
Pradeepa Jayachandran1, Elim Hong2, Rachel Brewster1 1Department of Biological Sciences, University of Maryland, Baltimore County 2Center for Neuroscience, Children's National Medical Center
instead of:
Pradeepa Jayachandran1, Elim Hong2, Rachel Brewster2 1Center for Neuroscience, Children's National Medical Center 2Department of Biological Sciences, University of Maryland Baltimore County

A correction to the author affiliations has been made for the article Labeling and Imaging Cells in the Zebrafish Hindbrain.

Erratum: Labeling and Imaging Cells in the Zebrafish Hindbrain

解决方案<ul><li>汉克的股票＃1：8.0 g氯化钠，氯化钾0.4克在100毫升DD H 2 O </li><li>汉克的联合＃2：0.358克钠2 HPO 4无水，0.60克KH 2 PO 4 100毫升DDH 2 O </li><li>汉克的股票＃4：0.72克氯化钙50毫升DDH 2 O 2 </li><li>汉克的股票5：1.23克硫酸镁 x7H 2 O在50毫升DD H 2 O </li><li> 1 + X磷酸盐缓冲液：0.8％氯化钠，0.02％氯化钾，0.02 M磷酸盐，pH值7.3 </li><li>封闭液：2％正常山羊血清，1％牛血清白蛋白（BSA），1％dimethysulfoxide（DMSO） </li></ul>

labeling and imaging cells in the zebrafish hindbrain

理解，塑造了早期脊椎动物胚胎的形态发生过程的关键是对高分辨率图像细胞的能力。在斑马鱼胚胎注射，镶嵌表达质粒DNA结果，使细胞的单细胞或小群的可视化<sup&gt; 1</sup&gt;。我们描述了如何注射质粒DNA编码一个无处不在的启动子控制下的膜有针对性的绿色荧光蛋白（mGFP）可用于神经胚细胞成像。这个协议的核心是节和实时高分辨率成像标记细胞的方法。该协议涉及mGFP的DNA注入到年轻的斑马鱼胚胎。胚胎，然后vibratome切片，抗体标记和共聚焦显微镜成像处理。另外，表达mGFP的活胚胎，可使用时间推移共聚焦显微镜成像。我们以前使用这种简单的方法来分析的驱动器在斑马鱼胚胎的后脑地区的神经管形成的细胞行为<sup&gt; 2</sup&gt;。允许前所未有的可视化细胞的形状和组织中的神经管，而这种方法使一个更好地了解在神经胚细胞动力学实时成像补充固定的准备。

Watch this Scientific Journal Video about Labeling and Imaging Cells in the Zebrafish Hindbrain at JoVE.com

Labeling and Imaging Cells in the Zebrafish Hindbrain

理解，塑造了早期胚胎的形态发生过程的关键是对高分辨率图像细胞的能力。我们在这里描述膜有针对性的绿色荧光蛋白标签在整个斑马鱼的胚胎细胞的单细胞或小群的技术。

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 ...

labeling-and-imaging-cells-in-the-zebrafish-hindbrain

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

11.3K 次观看.  University of Maryland, Baltimore County. 理解，塑造了早期胚胎的形态发生过程的关键是对高分辨率图像细胞的能力。我们在这里描述膜有针对性的绿色荧光蛋白标签在整个斑马鱼的胚胎细胞的单细胞或小群的技术。

视频: Labeling and Imaging Cells in the Zebrafish Hindbrain

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 ...

科研

神经科学

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

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 ...

The Mouse Hindbrain As a Model for Studying Embryonic Neurogenesis

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded forebrain neuroepithelium is not amenable to wholemount analysis to examine organ-wide neurogenesis patterns. Moreover, defining the mechanisms of forebrain neurogenesis is not necessarily predictive of neurogenesis in other parts of the brain; for example, due to the presence of forebrain-specific progenitor subtypes. The mouse hindbrain provides an alternative model for studying embryonic neurogenesis that is amenable to wholemount analysis, as well as tissue sections to observe the spatiotemporal distribution and behavior of neural progenitors. Moreover, it is easily dissected for other downstream applications, such as cell isolation or molecular biology analysis. As the mouse hindbrain can be readily analyzed in the vast number of cell lineage reporter and mutant mouse strains that have become available, it offers a powerful model for studying the cellular and molecular mechanisms of developmental neurogenesis in a mammalian organism. Here, we present a simple and quick method to use the mouse embryo hindbrain for analyzing mammalian neural progenitor cell (NPC) behavior in wholemount preparations and tissue sections.

This article demonstrates how the mouse embryonic hindbrain can be used as a model for studying developmental neurogenesis in both whole organ and tissue section preparations.

小鼠后脑作为研究胚胎神经再生的模型

The mouse embryo forebrain is the most commonly employed system for studying mammalian neurogenesis during development. However, the highly folded ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

用双光子激光消融神经种群及其对斑马鱼幼虫钙成像和行为记录的评价

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

幼虫斑马鱼外系头发细胞的活体钙成像

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

通过Sialoglycan的代谢标记识别主要神经干细胞和祖细胞的细胞表面标记

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

使用天然多酚姜黄素对脑组织淀粉样斑块进行标记和成像

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...