这项工作是由威康信托高级研究奖学金一，戴维斯支持。

1。 果蝇准备清扫前（据E. Gavis，普林斯顿大学） <ol><li>丢弃从一个人烟稀少接种瓶的成年人和转让一瓶25°C在实验之前。一旦新的成年人已经开始出现，等待两天。 </li><li>采取蝇小瓶含有固体食物大约10毫升添加0.5-0.7克干酵母（见附表），45度角。不飞食品用酵母覆盖整个表面。 </li><li>酵母，加水几滴足以水合物（图1A）。放置在45度的小瓶，让酵母约3分钟，浸泡水（图1B）。 </li></ol>注：此外，预混料1酵母粘贴在一个单独的容器中，并用锅铲添加粘贴到小瓶。 <ol start="4"><li>麻醉注射CO 2气体在瓶中的苍蝇。当他们停止移动，刀尖上的CO 2无意识的苍蝇</sUB&gt;排序（图1C）飞垫。 </li><li> 10-15女性在解剖范围和5所需的基因型的男性隔离用细尖的油漆刷（图1D）。男性存在claspers，一个黑暗的突出结构，在他们的后路，是明显的。 </li><li>选择苍蝇添加到yeasted瓶（图1E）。在25°C的地方为24-60小时。孵育短的时间内为更多的年轻％，中期阶段蛋商会和较长时期后期鸡蛋商会。 </li></ol> （2） 果蝇的卵巢解剖<ol><li>放置一个1.5号，22×50毫米的盖玻片（图2A）的卤烃油小滴。将一边准备盖滑，它不会的方式。 </li><li>麻醉注射到CO 2垫CO 2气体和小费在yeasted卑鄙的苍蝇。 </li><li>用锋利的细鼻钳把握养肥个别女性在腹部前（不与日é腹部和胸部）（图2B-C的）。 </li><li>女性用钳子举行，约2-3厘米以上的准备盖玻片，在解剖显微镜下查看。 </li><li>前仍然持有，使用的第二套钳消除极端的女性后方（图2D）的组织。注：肠组织通常会与后端拉。擦拭钳取出的组织。 </li><li>虽然仍保持前的女性，使用擦拭的镊子轻轻挤压或按摩腹部，前赶制的后路。按摩腹部，在同样的方式从管牙膏。 </li><li>从腹部和棒钳（图2E - F）月底将被推迟两个大的不透明结构。这些都是成对的卵巢。 </li><li>触摸到下面的幻灯片上的卤烃油的卵巢。 </li><li>重复剥离1-2次，给予4-6卵巢在盖玻片上的油（FigurË2G）。 </li></ol> 3。隔离卵巢管注：每个卵巢含有约16的6至10个鸡蛋像一个字符串10珠安排的不同阶段商会的卵巢管。 注：隔离卵巢管之前，调整解剖显微镜照明光源，使样品取得浅的角度。这使对比样品，让年轻的阶段蛋商会的可视化。 <ol><li>在石油，封闭钳（图3A）在后端（古戏台）通过消除连接组织分离的两个卵巢。 </li><li>东方权左侧和最年轻的阶段最古老的阶段，个体的卵巢。年轻的阶段，在卵巢卵商会是透明的，形成卵巢尖底。 </li><li>一双中占主导地位的手钳，轻轻抓住卵巢后，最好是由残余的日é结缔组织组织。按住钳发生卵巢，用解剖探头（或钨针），梳理出个别的卵巢管（图3B - D）。 </li></ol>注：个人卵巢管会之间的年轻阶段（germarium要举办10），打破旧的阶段是太大，无法从完整的卵巢中分离。 注：穿刺与解剖探头的后期阶段的卵母细胞，将导致入油泄漏的细胞质，使个别卵巢管的提取非常具有挑战性的。如果后期卵母细胞被刺破，移动到一个新的卵巢。 <ol start="4"><li>一旦卵巢管是免费的卵巢，用解剖的探头，将其拖动到油滴中心和实验所需的方向东方。注：油中的卵巢管会解决，并坚持以玻璃。 </li><li>重复这个过程中（3.3-3.4），直到有15-25个人的卵巢管。注：这可能需要解剖multip乐卵巢。整个过程中应少于15分钟。 </li></ol>注：最好是几个苍蝇，而不是减少苍蝇双侧卵巢解剖实验，以增加N多的苍蝇从解剖各一个卵巢。 注：粗糙处理的卵巢管会导致不健康的卵母细胞，可导致实验中的文物。 注：卵母细胞就会开始显示，由于强调40分钟后，从卵巢解剖（比较图7E对F）的表型变化。 <ol start="6"><li>一旦卵巢管，在适当的方向（图3E），用解剖的探头和封闭镊子移动的石油过剩的卵巢组织。这种材料可以从镊子上的纸巾擦了擦。 </li></ol> 4。隔离个别后期蛋商会（据E. Gavis，普林斯顿大学） <ol><li>与一双镊子在domin蚂蚁的手，抓住一个孤立的卵巢后，最好是由结缔组织的残余。虽然在地方的镊子卵巢，引入后端的卵巢附近的镊子（图4A）的解剖探头。避免后期蛋商会之间插入探头穿刺任何蛋室。 </li><li>转危为安卵巢解剖针，直到它退出在早期阶段蛋商会（图4B）。正确完成后，探测器将删除举行的卵巢管和后期蛋商会在地方的结缔组织。 </li><li>重复步骤4.2，直到盖玻片上的卵巢管蔓延。注：当卵子商会不再堆放在彼此顶部，这一步完成。 </li><li>使用解剖探头，后期卵母细胞分开，以便容易成像（图4C）。 </li></ol>注：穿刺卵母细胞，而解剖后期蛋商会是不是谴责与解剖我年轻的阶段ndividual卵巢管。 注：14阶段蛋商会，使用的镊子，把握方向背附属物。 5。注射剂注：对于中期阶段的卵母细胞注射，东方的卵巢管垂直于长轴的盖玻片。 <ol><li>打开成像显微镜[在我们的例子从应用精密DeltaVision核心和加载适当的成像设置（图5A-B的）。 </li><li>打开的注射器具（图5C）。 </li><li>加载装载尖（图5D-E），注射针头。被注入的解决方案应在高速离心简单，以避免存款，可能会阻止注射针。 </li><li>将装的注射针注射器。照顾，以避免破坏针（图5F）。 </li><li>协助畅通针，从伤口或破盖玻片1放置一个玻璃2X10毫米的小块油中含有的卵巢管下降的一个侧面。确保这个玻璃碎片被油覆盖。这种玻璃将成为打破或畅通针到RAM针尖对。 </li></ol> 6。注射荧光RNA 开始之前：建立注射装置和微型机械手的控制等，注射针头，在视野的中心位置。 <ol><li>装载的样品被注入显微镜舞台上。 </li><li>选择20X客观。也可用于更高或更低的放大率目标。 </li></ol>注：如果使用点访问功能自动化阶段，它是最好的，以纪念所有阶段开始前8-9注入卵母细胞。这允许容易访问和选定的卵母细胞的注射。 <ol start="3"><li>降低注射针，直到它触及油。中心在物镜的针尖。 </li><li>转关闭房间的灯打开亮场显微镜的光。选择标记点的名单上的第一点（卵母细胞）。通过目镜的同时，重点对卵母细胞（图6A）。 </li><li>手动降低针，直到它是在同一平面上的卵母细胞和可见光下的卵母细胞作为重点。 </li><li>使用机械手的控制，移动，直到它的卵母细胞内注射针点。往往是一个快速刺向运​​动较缓慢前进的成功。一旦进入，退出从体积小针和视觉的效果进行评估，需要重复。如果注入成功，将卵母细胞的细胞质内流离失所，如果这不是明显的重复。这是没有必要的材料，以确定是否已注入荧光图像。注射后，取出卵母细胞内（图6B-G）的针。 </li><li>注入流体的确切数额难以定量。为了增加注射量，佐剂ST的压力或注入脉冲的时间，打破了针尖略有增加开放或重复使用注射脉冲。 </li></ol>注意：整个注射过程中应采取正确执行时不到10秒。广泛刺伤或缠绵与针内卵母细胞，将导致严重损害蛋室。 <ol start="8"><li>在选择下一个显着的卵母细胞，提高高油注射针足够，以免联系其他的卵母细胞在旅行路径。 </li><li>在新的卵母细胞，重复步骤6.6。这样，直到所有所需的卵母细胞注入。 </li></ol>注：如果出现错误，而注射，移动到下一个标记的卵母细胞。不要浪费时间对受损的卵母细胞。 注：针注射会议期间可能会成为堵塞。在这种情况下，将毗邻的玻璃碎片在盖玻片上的油针。风趣h的玻璃，并在同一焦平面的针，轻轻公羊玻璃针（图6H）。测试针推注射按钮，并看到如果任何液体退出针尖畅通。 <ol start="10"><li>当所有的卵母细胞已注入，手动移动的针，出油和显微镜的光路方向出来。确保它是在一个安全的方向，以避免损伤眼睛。 </li></ol>注：如果长时间蛋室隔离和卵母细胞注射后，卵母细胞将难以注入并表现出与压力相关的表型变化。粗糙处理的卵母细胞或注射过剩体积（比较图6I，J，K），可能会出现类似的问题。 7。实时成像实验设计<ol><li>从列表中选择首先注入卵母细胞。测试注入卵母细胞内成像荧光的一帧（图6G）。 </li><li>配置实验参数，包括：激发波长，发射路径，激发时间，Z-Stack与集合之间的时间。注：欲了解更多这方面的资料，看到帕顿河，等。 （2010）2。 </li><li>收集的数据集。 </li></ol> 8。代表结果 在体外合成网站-546 GRK的RNA注射到Me31B :: GFP蛋腔（图7A）。 RNA的定位于背侧前的卵母细胞和细胞核周围形成了一个帽（图7B）。对于RNA的定位更多的例子，见麦克杜格尔N.等。 （2003年）11。 中期和后期卵母细胞表达荧光标记蛋白（TAU-GFP，图7C）或RNA标记系统（GRK * mCherry，图7D）可成像没有注射。 <img alt="图1" src="/files/ftp_upload/3679/3679fig1.jpg" /> 图1。 <img alt="图2" src="/files/ftp_upload/3679/3679fig2.jpg" /> 图2。 <img alt="图3" src="/files/ftp_upload/3679/3679fig3.jpg" /> 图3。 <img alt="图4" src="/files/ftp_upload/3679/3679fig4.jpg" /> 图4。 <img alt="图5" src="/files/ftp_upload/3679/3679fig5.jpg" /> 图5。 <img alt="图6" src="/files/ftp_upload/3679/3679fig6.jpg" /> 图6。 <img alt="图7" src="/files/ftp_upload/3679/3679fig7.jpg" /> 图7。

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<html>活细胞成像是一个强大的研究细胞过程中实时检测。除了简单明亮的实地观察，除了利益的蛋白质和RNA的荧光标记，导致许多突破。在这里，我们提出了一个成像的个人生活可以结合利用卵母细胞与遗传和生化检测的协议。 在这个协议中，我们也解释如何生活的卵母细胞注射实验操作。有许多材料注入的可能性，包括在体外合成荧光标记的RNA二级结构的能力，以检测RNA的直接定位（Ball和戴维斯，未发表）和抗体，抑制蛋白质的功能6。今后的工作中可能会看到其他标签组件的引进和蜂窝机械生活的卵母细胞，使分子机制进行测试。 T“保活细胞组织的活力和健康是必不可少的工作。在这个协议中，我们指出了一些步骤，可导致蛋室的压力。例如，尽管石油是优越的成像，延长石油文化可以导致蛋室强调，这可以很容易地在明亮的领域暴露监督检查核的形态和位置，卵母细胞的细胞膜会扭曲和压力下泡（比较图7E（重读）Figure7F（强调））第一阶段9个蛋商会表明RNA的定位和边界细胞迁移的12多个小时。然而，7/8阶段蛋商会将被删除卵巢约40分钟后开始表现出晕碳油的不良影响女性。晚期蛋商会，阶段11日至14日，可以正常发展，因为从这些阶段在13毛囊细胞分泌蛋壳要么油或水介质中。它已被回购rted，除了水的昆虫媒介的胰岛素可以维持长达6小时14和germaria 14小时15阶段9卵母细胞。在所有情况下，积极机动的蛋室应避免，因为它强调的卵母细胞，并减少其可行性。成像少蛋室和照顾对待每一个轻轻的最佳方式是，以确保最佳的可行性。

夹层中使用的工具应该是干净的，但不需要进行高压灭菌。擦拭的ETOH工具和让他们干，然后再开始。 <table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>细尖油漆刷 （艺术家紫貂，圆形，大小为00或0） </td><td>可从大多数美术用品网点</td><td></td><td>重要的是质量好刷</td></tr><tr><td>卤烃产品公司，95系列</td><td>卤烃产品公司，887 Kinderkamack路，河边NJ 07661 </td><td></td><td>欧洲经销商： solvadis（GMBH的） 一定冒泡通过油的空气含氧。 </td></tr><tr><td>盖玻片没有。 1.5，22 x 50毫米</td><td>国际1号，厂商VWR猫= 631-0137-22-50MM </td><td>门泽尔，格拉泽22-40MM MNJ的400-070T </td><td></td></tr><tr><td>杜蒙5号 - Dumostar </td><td>罚款科学工具</td><td> 11253-20 </td><td>的“Biologie”小费也不错但更脆弱</td></tr><tr><td>解剖探头</td><td>罚款科学工具</td><td> 10140-01 </td><td></td></tr><tr><td>解剖显微镜</td><td>奥林巴斯</td><td> SZ61 </td><td></td></tr><tr><td>二氧化碳垫</td><td>可通过 <a href="http://www.flystuff.com">http://www.flystuff.com</a> / （分工杰纳西科学） </td><td> 59-114 （789060 Dutscher） </td><td>欧洲经销商： 的COM</a> / &gt; Dutscher <a href="http://www.dutscherscientific.com">WWW。dutscherscientific的 。</a> 它也比较容易定制建立你自己的飞垫</td></tr><tr><td> KimCare </td><td> kimbery克拉克KimCare医疗湿巾</td><td> KLEW3020 </td><td></td></tr><tr><td>显微镜DeltaVision </td><td>应用精密</td><td> DC-CORE中</td><td></td></tr><tr><td>微操作机器人</td><td>伯利显微操纵仪 流明动力集团有限公司。 2260 Argentia路，米西索加，安大略省，加拿大L5N 6H7 </td><td>伯利PCS-5000系列，TS-5000-300 </td><td> <a href="http://www.ldgi-burleigh.com">http://www.ldgi-burleigh.com</a> /（原EXFO） </td></tr><tr><td>注射器具</td><td>三技术研究公司 2961退伍军人大道 洛杉矶，加州90064 </td><td> MINJ-1 </td><td>与修改，以持有人的Eppendorf Femptotips </td></tr><tr><td>注射针头</td><td> Eppendorf公司无菌Femtotips I和II </td><td> 930000043 </td><td>不同的技巧，更好地为不同的应用</td></tr><tr><td>载入提示20μL </td><td> Eppendorf公司</td><td> 5242956.003 </td><td></td></tr><tr><td>干酵母</td><td>弗莱希曼的酵母</td><td> ＃2192 </td><td></td></tr></tbody></table>

preparing individual drosophila egg chambers for live imaging

活细胞成像是一个重要的技术应用到一些果蝇作为模型来探讨，如轴规格，细胞分化和器官1组织。实验样品的正确准备是至关重要的，往往被忽视，步骤。编制的目标是确保相关生理和建立最佳的成像条件。为了保持组织的活力，这是关键，以避免脱水，缺氧，过热或中等恶化2。 果蝇的卵室是为建立良好的制度，研究有关的问题，而不是有限的，身体图案，基因定位和细胞骨架组织3,4。对于早期和中期阶段的蛋室，安装在卤烃油是良好的生存，它允许氧的自由扩散，防止脱水和缺氧，具有精湛的显微镜的光学性质。 IM通过引进转基因蛋室或物理注射标记的RNA，蛋白质或抗体5-7荧光蛋白老化是可能的。例如，除了对MS2的动物的基因组结构，以使在卵母细胞8 mRNA的实时观察。这些结构允许在体内的基因标记，通过利用其外壳蛋白9的MS2噬菌体RNA茎环互动。 在这里，我们提出了一种提取卵巢，以及雌性果蝇隔离个别卵巢管和蛋商会的协议。详细描述了果蝇卵子见艾伦C. Spradling（1993年，重印2009）10。

Watch this Scientific Journal Video about Preparing Individual Drosophila Egg Chambers for Live Imaging at JoVE.com

Preparing Individual Drosophila Egg Chambers for Live Imaging

“<em&gt;果蝇</em&gt;蛋商会是一个很好的模型，为研究基因定位机制。为了捕捉支持本地化进程的动态事件，活组织快速高分辨率成像是必需的。在这里，我们提出了以最小的中断现场样品的解剖和成像协议。

准备个人果蝇蛋钱伯斯实时成像

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

preparing-individual-drosophila-egg-chambers-for-live-imaging

Research

JoVE Journal

Biology

Preparing Individual Drosophila Egg Chambers for Live Imaging

19.1K 次观看.  University of Oxford. “果蝇蛋商会是一个很好的模型，为研究基因定位机制。为了捕捉支持本地化进程的动态事件，活组织快速高分辨率成像是必需的。在这里，我们提出了以最小的中断现场样品的解剖和成像协议。

视频: Preparing Individual Drosophila Egg Chambers for Live Imaging

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

住在果蝇眼睛成虫光盘胶质细胞迁移成像

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

科研

生物学

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

编制果蝇实时成像的胚胎使用悬滴协议

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

收获和准备果蝇胚胎的电生理记录和其他程序

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

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 ...

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 ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

GFP标记的蛋白质在实时成像<em&gt;果蝇</em&gt;卵母细胞

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

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  ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

番茄/ GFP-FLP / FRT方法马赛克成人的实时成像<em&gt;果蝇</em&gt;感光细胞

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

可视化和跟踪活果蝇黑色素细胞卵室的内源性 mRNA

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...