罗森布拉特实验室的科学讨论，建议和意见，我们感谢成员。我们还要感谢玛丽哈洛伦请提供质粒RFP - UtrCH和大卫格伦沃尔德提供的CK：GFP转基因斑马鱼。还要感谢格蕾琴国王和良好的维护和斑马鱼的护理工作人员在犹他州立大学的集中斑马鱼资源。这项工作是由美国国立卫生研究院NIGMS美国国立卫生研究院主任的新的创新奖1 DP2 OD002056 - 01至JR支持。 GTE公司是由美国国立卫生研究院癌症多学科培训项目资助5T32 CA03247 - 8的支持。

在发展中国家斑马鱼的表皮细胞挤压在肌动蛋白动力学的可视化的基本工作流程发展中国家斑马鱼的表皮是由两个不同的层，面层（或周皮）和基底层的细胞接触基底膜 2 。外表面层的细胞发生凋亡，并从组织消除挤出 3（图1） 。为了实时可视化这个过程中，我们注入的RNA编码红色荧光蛋白融合成单细胞阶段的转基因表达绿色荧光蛋白（GFP斑马鱼utrophin，肌动蛋白结合蛋白（RFP - UtrCH）4,5，调宁蛋白同源结构域）根据分层的上皮细胞启动子细胞角蛋白8月6 日（图2A） 。虽然在后的RNA注射的动物表达RFP - UtrCH是无处不在，我们集中表达RFP和GFP遵循的表皮（图2B）中的肌动蛋白动力学专上肤浅的表皮细胞。然后，我们把与G418幼虫诱导细胞凋亡的挤压和图像使用一个旋转盘共聚焦显微镜的表皮和微丝，并获得4D数据集。 实验开始之前<ol><li>使用mMessage mMachine SP6试剂盒（Ambion公司）从一个线性DNA模板体外转录RNA和净化使用的RNeasy试剂盒（QIAGEN）的上限核糖核酸。使用无菌水稀释的RNA〜60ng/ul，直到注射35μL分装并储存于-80 ° C。注意：避免重复冻结/解冻，以防止RNA的降解。 </li><li>制作模具在E3胚胎培养基（E3）的2％琼脂糖40ML浇筑100 × 15mm的培养板，此托盘放置在一个显微注射模具（自适应科学工具，＃I - 34）期间举行注射胚胎。一旦琼脂糖凝固后，取出模具，暴露在琼脂糖槽。储存在4 ° C，直到准备使用该板块。 </li><li>拉细丝（0.78外径1.0MM，编号，10公分长）和一个萨特的P - 97在以下设置（485，热拉50，速度60，延时/ 90，压力200移液器拖轮使用注射硼硅玻璃毛细管针）。注：不同类型的玻璃，将规定的上市条件的优化。 </li><li> 1％低熔点（LM）的琼脂糖在E3和存储毫升分装在42 ° C。要小心，因为从股票的E3蒸发可以增加琼脂糖浓度。 </li><li>保持标准的实验室条件下的成年斑马鱼，一个普通的光/暗周期14小时光照和10小时黑暗的 7 。前一天晚上的实验设置分频器分离坦克3名女性和2名男性，鱼交配。第二天早晨，改变水箱中的水和拉分压器，当灯光来。 </li></ol> 1。注射荧光标记的肌动蛋白结合蛋白编码RNA的<ol><li>在冰上解冻的RNA。 〜30ng/μl最终的RNA浓度为1:1的比例加入0.5％酚红的RNA装入一把拉住毛细管针回填加入1μl的解决方案。 </li><li>收集〜75 1 - 8细胞阶段CK：在E3 GFP的胚胎。吸管收集5胚胎的显微注射模具¾巴斯德吸管轻轻东方在与福斯特杜蒙＃5镊子的低谷胚胎。注意：小心迅速开展工作，以避免注射到多细胞阶段的胚胎，这可能会导致镶嵌注入的RNA表达。 </li><li>一个标准的实验室解剖立体显微镜下，使用压力控制微量（哈佛大学器械的PLI - 100的Pico -注射器）胚胎的卵黄注入的RNA。红点（酚红）应该在胚胎注射后可见。每个实验注入至少50个胚胎。 （见以前的Jove协议一个详细的RNA注射到斑马鱼的胚胎9协议）。注意： <ol type="A"><li> 〜2nl卷应使用的RNA注射到细胞阶段的胚胎。要确定注入的RNA的量，分配到校准微米幻灯片上的矿物油下降或比例尺内置目镜显微镜下的RNA丸。 RNA的液滴应该是一个完美的球体。计算所使用的方程交付量：卷（NL）=4/3πr3。调整通过改变喷油压力或时间上的仪器的交付量。 </li><li>也应采取注入RNA的最低数额，允许可视化的荧光，高浓度的RNA可以到发育中的胚胎有毒。要确定适当的表达水平，实验剂量曲线应进行运行实验之前&lt;/ LI&gt; </ol></li><li>胚胎进行排序，以消除任何未受精或损坏的鸡蛋和地方所有剩余的胚胎在28.5 ° C。 </li><li> 24小时后，使用荧光解剖显微镜选择，有一个高水平的绿色荧光蛋白（表皮）和RFP（肌动蛋白）荧光的斑马鱼胚胎。放置在选定的胚胎28.5 ° C，直到准备进行诱导细胞凋亡的药物治疗。 </li></ol> 2。在使用G418的斑马鱼幼虫诱导细胞挤出我们已经发现，暴露的氨基糖甙类抗生素G418的，或Geneticin，导致由一个未知的机制在发展中国家斑马鱼幼虫3的表皮细胞凋亡和挤压。更重要的是，这种治疗方法只幼虫，后4天施肥和老年人。我们发现，可以发现在任何给定的G418治疗幼虫斑马鱼的鳍上皮的时间挤出〜5-25细胞。由于挤出凋亡细胞的数量可能有所不同从鱼到渔，我们建议安装多种鱼类成像。 <ol><li>收集和地点4天的受精后（DPF）35 x 10mm的文化包含3毫升的E3（MLS）的菜都将参展GFP和RFP荧光的斑马鱼幼虫。可以治疗25幼虫在这种规模的培养皿。 </li><li>仔细取代含有1mg/ml的G418和孵化幼虫在药物4小时的E3 3mLs媒体在28.5 ° C需要注意的是G418的对光敏感，所以要小心，保持覆盖的培养皿或在黑暗中。 </li><li>删除媒体和预热28.5 ° C E3的媒体含有0.02％Tricaine的更换，并进行安装的幼虫。 </li></ol> 3。安装成像斑马鱼幼虫<ol><li> 3幼虫转移到1.5 mL Eppendorf管和后取出鱼沉到试管底部的E3。我们发现，3动物可以正确安装的琼脂糖凝固之前。 </li><li>在盖玻片上一个MatTek培养皿的底部使用一个吸管尖切进管，吸管1％LM -琼脂糖（42℃）120ul，混匀，然后轻轻吸管幼虫和琼脂糖混合物。 </li><li>使用插入针或钨针，快速定位的幼虫，所以他们对MatTek盘的玻璃罩平直，一个FST的针座。 注：我们通常使用直针第一东方幼虫，然后一个L形的针，以确保他们对玻璃罩持平，而防止损坏的标本。 </li><li>一旦琼脂糖凝固后，轻轻地填写E3含0.02％Tricaine的菜。注：另外，G418可以添加到成像盘继续诱导挤压E3，虽然长期暴露1m​​g/ml的G418的杀幼虫。 </li></ol> 4。住在细胞挤出肌动蛋白动力学和个人的上皮细胞行为的成像，使用一个旋转的光盘共聚焦显微镜我们发现，从发展的斑马鱼幼虫的表皮细胞凋亡挤出的整个过程大约需要20分钟3。在这里，我们将演示如何设置一个时间推移的成像实验，收集通过对斑马鱼表皮的单层一系列z平面可视化挤出过程。我们发现，造成重大典型的宽视场荧光显微镜的光漂白微丝和不允许timelapse成像。因此，最大限度地发挥我们的决议，并防止漂白，我们使用一个旋转盘共聚焦显微镜。下面我们介绍如何设置使用安道尔IQ软件与尼康显微镜的实验，虽然讨论的原则可以应用到类似显微镜调校。 <ol><li>首先，打开氩（488nm GFP的激发）和氦氖（RFP的激发561nm）激光器，显微镜，照相机和啶灯。 </li><li>在安道尔IQ软件（版本1.10.1），创建时间的一系列协议，其中包含你想要的波长图像，图像捕获和次数之间的间隔的频率应重复捕捉。 </li><li>轻轻将Ma​​tTek菜含有您的标本，在显微镜舞台上，并使用透射光20倍的目标，把重点放在幼虫。 </li><li>移动到正确位置的40倍的水浸泡目标（NA 0.8）。使用这个目标，我们可以拍摄约20％至25场，这让我们跟随在挤出过程中的细胞行为的同时，也解决了亚细胞的肌动蛋白动力学细胞。注意：相同的安装和成像策略可应用于直立显微镜，虽然40倍的水浸泡长工作距离的目标，必须使用。 </li><li>小心地40X目标上方的水下降，并迅速MatTek菜放在上面。不E：蔡司Immersol浸泡的解决方案也可用于水的蒸发是一个问题。 </li><li>确定使用阶段或DIC照明的标本，然后用488nm epifluorescence专门专注于GFP阳性表皮。 </li><li>切换到共焦扫描模式。调节激光的强度和曝光时间，为每个通道（488nm和561nm）作相应。照顾，平衡信号的最佳检测激光功率和曝光时间，并防止任何漂白或毒性。 </li><li>要确定挤出细胞，使用epifluorescence的可视化的绿色荧光蛋白标记的表皮和识别的细胞组中的一个经典的花环图案，中间一个小圆形细胞周围的细胞集合组成。使用共焦扫描模式，也应该有一个积累RFP标记的肌动蛋白作为一个环在中间，周围的小圆形细胞的细胞挤出的一个标志可见。一旦你已经确定了一个挤出细胞，迅速成立显微镜获取4D共聚焦数据集（XYZ时间）。 </li><li>设置在z平面的上部和下部的点看到一个单层表皮，包括挤出细胞，然后选择步长的大小。对于时间推移成像，图像采集和重复次数的时间间隔。例如，我们通常会获得一个Z系列横跨5 - 10微米，1微米的步长，每隔1-2分钟，30分钟。 </li><li>要查看数据，我们通常使Z系列的3 - D的预测，并保存为影片文件是兼容快速时间（苹果）设置的数据。在这种方式下，我们可以可视化的肌动蛋白环，随着时间的推移而死亡细胞周围发生的上皮行为的收缩。 </li></ol> 5。代表性的成果<img src="/files/ftp_upload/2689/2689fig1.jpg" alt="图1"/> 图1。细胞凋亡挤出示意图。微丝显示为红色，GFP阳性表皮细胞都是绿色的。 <img src="/files/ftp_upload/2689/2689fig2.jpg" alt="图2"/> 图2。实验工作流程。 A.实验的工作流程示意图， B。 RFP - UtrCH表达一个4dpf长江：绿色荧光斑马鱼的表皮。 <img src="/files/ftp_upload/2689/2689fig3.jpg" alt="图3"/> 图3。帧从一个时间推移电影，活肌动蛋白动力学在上皮细胞挤出 ​​的过程如下（Z -预测） 。箭头表示超过2分钟的过程中，合同和关闭邻近的细胞所形成的肌动蛋白环。

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动物福利保障在此使用的斑马鱼， 斑马鱼的协议执行的所有程序，已批准的机构动物护理和使用委员会（IACUC）在美国犹他州大学（动物福利保证＃10-07017） 。

这里所描述的协议演示了一个简单而直接的方法来可视化挤压过程，在现场的上皮组织。这种类型的实验使我们以前没有固定组织分析赞赏的肌动蛋白动力学研究中的细微之处，因此，常见的免疫方法的补充。我们可以利用这个协议在组合化学抑制剂或基因突变体，以便更好地分析删除和清除细胞凋亡，我们期望与故障相关的挤压过程和研究疾病状态下会导致炎症反应或受损细胞的积累，为见于?...

<table border="1"><tbody><tr><th>名称</th><th>类型</th><th>公司</th><th>产品编号</th><th>评论</th></tr><tr><td>的RNeasy迷你包</td><td>试剂</td><td> Qiagen公司</td><td> 74104 </td><td></td></tr><tr><td> mMessage mMachine SP6套件</td><td>试剂</td><td> Ambion公司</td><td> AM1340 </td><td></td></tr><tr><td>隧道坦克</td><td>工具</td><td> Thoren水产系统</td><td></td><td></td></tr><tr><td>吸管泵</td><td>工具</td><td>贝尔艺术产品</td><td> F3789 </td><td></td></tr><tr><td> 5 ¾巴斯德吸取</td><td>工具</td><td> VWR </td><td> 14672-608 </td><td></td></tr><tr><td>显微注射模具</td><td>工具</td><td>自适应科学工具</td><td>我34 </td><td></td></tr><tr><td> 100x15mm文化板</td><td>工具</td><td>费舍尔</td><td> 08-757-12 </td><td></td></tr><tr><td> Flamming /布朗Micropipet拖轮</td><td>工具</td><td>萨特仪器公司</td><td>型号P97 </td><td></td></tr><tr><td>与长丝的高硼硅玻璃毛细管</td><td>工具</td><td>萨特仪器公司</td><td> B1400 - 78 - 10 </td><td>外径1.0MM，编号0.78毫米，10公分长</td></tr><tr><td>电极储存罐</td><td>工具</td><td>世界精密仪器</td><td> E210 </td><td></td></tr><tr><td>酚红</td><td>试剂</td><td>西格玛</td><td> P0290 </td><td>在DPBS 0.5％ </td></tr><tr><td>压力控制的微量及微操作机器人</td><td>工具</td><td>哈佛器械</td><td> PLI - 100 </td><td></td></tr><tr><td> 35x10mm文化菜</td><td>工具</td><td>蜂星</td><td> 627-160 </td><td></td></tr><tr><td> G418（Geneticin） </td><td>试剂</td><td> GIBCO公司</td><td> 10131-035 </td><td> 50毫克/毫升，在卫生署 2 0 </td></tr><tr><td>杜蒙＃5镊子</td><td>工具</td><td>精细科学的工具</td><td> 11253-20 </td><td></td></tr><tr><td> 12厘米引脚持有人</td><td>工具</td><td>精细科学的工具</td><td> 26016-12 </td><td></td></tr><tr><td>插入销</td><td>工具</td><td>精细科学的工具</td><td> 26007-02和03 </td><td></td></tr><tr><td>玻璃底培养皿1.5玻璃罩</td><td>工具</td><td> MatTek </td><td> P35G - 1.5 - 10 - C </td><td></td></tr><tr><td>低熔体琼脂糖</td><td>试剂</td><td>费舍尔</td><td> BP1360 - 100 </td><td></td></tr><tr><td> Tricaine </td><td>试剂</td><td>西格玛</td><td> A - 5040 </td><td></td></tr><tr><td>解剖显微镜</td><td>显微镜</td><td>徕卡</td><td> MZ6 </td><td></td></tr><tr><td>解剖显微镜</td><td>显微镜</td><td>尼康</td><td> SMZ645 </td><tD&gt; </td></tr><tr><td>荧光解剖显微镜</td><td>显微镜</td><td>奥林巴斯</td><td> S2X12 </td><td></td></tr><tr><td>旋转的光盘共聚焦显微镜</td><td>显微镜</td><td>尼康</td><td> Eclipse的钛</td><td>配备一个Yokagawa转盘头</td></tr><tr><td> CCD相机</td><td>相机</td><td>安道​​尔</td><td> DV - 885 - VP </td><td> 1002x1004x8微米的正方形像素</td></tr></tbody></table>

live imaging of cell extrusion from the epidermis of developing zebrafish

稳态维持上皮组织需要不断清除而不破坏的屏障功能受损的细胞。我们的研究发现，死亡的细胞信号传递给他们住邻居的形式和合同的肌动蛋白和肌球蛋白环弹出上皮表，而关闭任何可能导致其退出的差距，这个过程被称为细胞挤出​​1。发展中国家斑马鱼的光学清晰度提供了一个极好的系统，以可视化挤出生活上皮。在这里，我们描述了一种方法来诱导和幼虫斑马鱼表皮的形象挤出。可视化挤出，注射F -肌动蛋白红色荧光蛋白标记的探针进入细胞阶段在表皮表达绿色荧光蛋白的转基因斑马鱼的胚胎和诱导凋亡的G418除了幼虫。然后，我们使用一个旋转的光盘共聚焦显微镜成像技术观察在细胞凋亡挤出过程中的肌动蛋白动力学和上皮细胞行为的时间推移。这种方法使我们能够调查上皮细胞在活的挤压过程中，将提供一个途径研究由故障消除细胞凋亡引起的疾病状态。

Watch this Scientific Journal Video about Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish at JoVE.com

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

死亡的细胞被挤压协调一致的收缩邻近的细胞，上皮组织，而不破坏的屏障功能。发展中国家斑马鱼的光学清晰度提供了一个极好的系统，以可视化挤出生活上皮。在这里，我们描述的方法来诱导和幼虫斑马鱼的表皮细胞决议形象挤压。

发展中国家斑马鱼的表皮细胞挤压实时成像

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

live-imaging-cell-extrusion-from-epidermis-developing

Research

JoVE Journal

Biology

14.6K 次观看.  University of Utah. 死亡的细胞被挤压协调一致的收缩邻近的细胞，上皮组织，而不破坏的屏障功能。发展中国家斑马鱼的光学清晰度提供了一个极好的系统，以可视化挤出生活上皮。在这里，我们描述的方法来诱导和幼虫斑马鱼的表皮细胞决议形象挤压。

视频: Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

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.  ...

科研

生物学

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

剖析器官的成年斑马鱼

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

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 ...

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 ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

发展和成人的制备果蝇大脑和视网膜实时成像

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

活细胞成像早期自噬活动：Omegasomes与超越

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.