这项工作是由陆国际律师事务所奖学金HY支持。 H.，一个简棺童车纪念医学研究的博士后研究基金RJJ，美国国立卫生研究院资助F32EY016309到DV，纽约州立大学的教务长的论文奖学金DJ，NIH GrantR01 EY13010到CD和一个DFG奖学金JR（RI 2208/1- 1）。我们感谢龚如心Vogt和帕梅拉Boodram的手稿上的评论。

1。介绍在这段视频中，我们将介绍三个定义的发育时期的视网膜解剖和免疫组织化学的方法：三龄幼虫，midpupal和成人阶段。虽然我们的协议也适用于其他蛹的阶段（早期阶段的详细信息，请参阅16），我们选择了midpupal的阶段，因为它是最佳的成像PR在一个焦平面，其核很容易识别，方便的可视化转录因子的表达模式。后期蛹视网膜剥离是非常相似的的成人视网膜剥离的。 首先，我们展示了如何收集幼虫和蛹在需要的阶段。然后，我们将解释如何获得最佳的解剖幼虫17日至18日 ，midpupal和成人视网膜。第二，我们展示了如何理解PR在这些阶段，采用免疫组织化学和激光共聚焦显微镜的发展。 昆虫的发育时期是温度依赖的。为了促进再生分期幼虫和蛹，我们建议提高都飞股票在25°C下一个12 hr/12小时，光/暗周期。 三龄幼虫产卵后约96小时（25°C），晚三龄幼虫停止摄食，漫步在墙壁上的食品瓶，在那里他们最终化蛹。使用软刷或镊子轻轻取出一个流浪的第三龄幼虫从墙上的食品瓶。 50％的蛹（“midpupae）约48小时后观察化蛹。对于确切的时间，你可以圆刚形成的白色蛹与食品瓶上的标记。或者，将所有白色的蛹获得一个新的小瓶一小瓶均匀分期（分期蛹的形态标准，请参阅19）。 48小时后，仔细REM从小瓶奥雅纳的midpupae，使用的钳子。 成虫：在CO 2焊垫的麻醉年轻的成年果蝇（羽化后2-5天）。使用镊子取出头，并将其储存在一个玻璃皿冷1X PBS。 3。解剖过程（大约1-2小时，每次实验） 收集标本后，将它们放置在Sylgard的解剖盘上一个冷PBS下降。每型约10-15视网膜解剖。 请参阅材料安全数据表以及机构的环境，健康和安全办公室的试剂和设备的妥善处理（见表结束时本协议）。 1。幼虫眼成虫盘<ol><li>定位前端的幼虫，其中包含了口钩。使用钳子Sylgard的菜的基础上，轻轻地按中间部分的幼虫。使用另一个呃钳抢口钩，拉轻轻地，慢慢远离身体。丢弃的主体的主要部分的，并使用口器持有的前部，以去除多余的组织，保持连接到大脑从成虫盘。 </li><li>大脑与成虫盘传输1X PBS三以及玻璃盘钳子抓住了嘴钩。执行固定步骤（步骤4.1）。 </li><li>固定后，取出唾液腺，脂肪组织和远离拉大脑，眼触角成虫盘。为了改善可访问性“困难”抗体，peripodial膜，它位于的表面上的上皮细胞，可以除去通过按住触角的盘的一部分和与解剖销剥离膜。接下来，进行免疫组织化学（步骤5.1）。 </li></ol> 2。 50％的化蛹/ Midpupal视网膜<ol><li>按住后端蛹的情况下，用钳子。蛹的情况下轻轻地取出前表面通过抓的角质层与其他镊子从puparium，慢慢地拉开了距离。剥离残留表皮，露出头。 </li><li>使用镊子提示刺穿的基础，软袋背前区。这提供了访问的头部。与P20吸管慢慢地吸了头，轻轻分开的头胸部。到冷鲜1X PBS驱逐头移液器吸头，去除多余的组织;抢头角质层，去除长鼻。继续固定步骤（步骤4.1）。 </li><li>皮尔斯视叶一个稳定的组织解剖针。将其他解剖针之间的层和视网膜分离的视网膜层/视叶。进行免疫组化（步骤5.1）。 </li></ol> 3。成人眼<ol><li>抓住中心，钳子和删除后头壳口器（喙），脂肪TISSUE和气管。 </li><li>用一个镊子，从脑组织中分离的眼睛，轻轻拉动侧身抢背头角质层。在大多数情况下，薄片，薄且透明的护套的脑的神经纤维，将保持附着到视网膜。这是比较容易除去叶片固定后。 </li><li>去除死皮的眼缘，但背后留下一小片，视网膜转移，以方便不同的解决方案。执行固定（步骤4.1）。 </li><li>将椎板一侧钳子抓住它，和其他镊子轻轻侧身拉不损害视网膜。清除残留的角质层，在安装过程中，因为它可以折叠到视网膜上。这两个步骤是至关重​​要的，否则将被遮蔽的叶片，保持角质层在成像过程中的光感受器，可视化。继续进行免疫组化（步骤5.1）。 </li></ol> 4。固定和天鹰毫微克（约1小时） <ol><li>存储解剖视网膜在1x PBS，放置在冰中，而新鲜制备的固色剂（稀释40微升37％的甲醛溶液，用360μl的1×PBS中，在冰上的涡流和存储）中的3孔的玻璃皿。我们建议继续与固定协议和冰（15分钟），以避免长期储存时间。 </li><li>更换1倍的PBS，用200μl3.7％的甲醛溶液。确保组织被淹没在固色剂。这是非常重要的，因为它可能无法被正确固定。如果有必要，去除气泡和气管的镊子。 </li><li>混和器中研磨15分钟，在室温下孵育。 </li><li>更换固色剂用1×PBS，用1×PBS冲洗两次。继续在PBS中洗涤30分钟（振动筛，在室温下）。 </li><li>更换PBS与PBST。用PBST冲洗两倍。 </li><li>继续与删除的peripodial的保护膜，幼虫眼盘（3.1.3）和纹层从midpupal /成人视网膜&lt;强&gt;（3.2.3，3.3.4）。 </li></ol> 5。免疫组化<ol><li>阻断：替换在PBST中并孵育视网膜上摇动器在室温下20分钟的5％正常血清用200μl的PBS。 </li><li>更换阻断溶液与初级抗体溶液。用封口膜密封的三个阱培养皿，并在摇动器在室温下孵育过夜。 </li><li>更换溶液用PBST冲洗3次。继续用PBST洗涤为在室温下至少1小时摇床。 </li><li>转移的二次抗体溶液和石蜡膜密封的玻璃皿上摇动器在室温下过夜孵育。 </li><li>替换的抗体溶液，用PBST冲洗三次。继续用PBST洗涤为在室温下至少1小时摇床。可将样本保持在PBST数天，但它是至关重要的，添加新鲜的PBST，每天至少一次，以防止它们干燥。 </li></ol> 6。中号ounting <ol><li>用于安装幼虫和midpupal的视网膜中 ，使用的P20视网膜转移到干净的载玻片上的中心。删除多余的PBST。使用钳子对准视网膜，以促进成像。 </li><li>加入10μl视网膜上安装介质。轻轻地用一个24X40-1的盖玻片和密封与透明指甲油覆盖它们。 </li><li> 成人视网膜安装，准备“桥梁”20：添加两个5微升滴50％甘油的玻璃侧的两端，并覆盖22X22-1盖玻片（0.13至0.17毫米厚）。 </li><li>与P20吸管取出PBST;，确保视网膜干不出来。加入20μl的良好的安装介质。使用安装介质转移视网膜的P20的“桥梁”的载玻片上。 </li><li>使用钳子东方（镜头要面对的载玻片上）和调整为了方便成像视网膜。如果有必要，去除气泡的P20。 </li><li>慢慢地地方24X40-1盖玻片（0.13至0.17毫米厚），从一个侧面顶部和密封边与透明指甲油或透明胶带。店内样品在显微镜载片上保护盒在4°C（保持在黑暗中）。 </li></ol> 7。代表性的成果本协议的应用程序作为一个例子，我们显示出有代表性的三个发展阶段，在视频可视化光感受器分化的结果。甲幼虫眼触角的圆盘和一个midpupal的的视网膜染色标记不同的感光器类型的抗体，在他们的发展（ 图1A，B）和两个视紫红质抗体染色成人视网膜感光亚型（ 图1C）可视化。

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没有利益冲突的声明。

1。故障排除根据我们的经验，解剖需要实践（可长达数周），并促进实现舒适的手位21搁在桌子上，肘部和前臂和手指接触的解剖盘。这样一来，只有拇指，食指和中指进行细微的动作。 卸下椎板不损坏感光体的情况下可能是最具挑战性的步骤。红眼野生型的实践与飞白眼突变体解剖，第一层是对红色的眼睛色素沉着更容易看到。此外，使用灯光?...

<table border="1"><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td>磷酸盐缓冲盐水（PBS1x，pH值7.4） </td><td>西格玛</td><td></td><td>准备10倍原液20。用蒸馏水稀释，得到1×PBS中并储存在室温下。酷上冰前解剖。 </td></tr><tr><td>的Triton-X 100的</td><td>西格玛</td><td> 9002-93-1 </td><td> 注意：刺激！戴上手套。用0.3％的Triton X-100（PBST）。准备50毫升1xPBS在室温下储存。 </td></tr><tr><td> 37％甲醛溶液</td><td> Fisher Scientific则</td><td> F75P1GAL </td><td> 注意：有毒的，可能的人类致癌物！戴上手套。固定步骤之前，刚准备3.7％的溶液，用PBS稀释的化学通风柜，储存在冰。 </td></tr><tr><td> 5％正常马血清</td><td>杰克逊免疫研究</td><td> 008-000-001 </td><td>准备5％体积/体积在PBST稀释。四度储存。 </td></tr><tr><td>小学和中学抗体（ 如驴抗羊的Alexa Fluor 488，驴抗兔的Alexa Fluor 555，驴抗小鼠的Alexa Fluor 647） </td><td> Invitrogen公司的分子探针</td><td> A11015 A31572 A31571 </td><td>稀释的二抗1:800 PBST和存储四度。 </td></tr><tr><td>的Alexa Fluor 488鬼笔环肽</td><td></td><td> A12379 </td><td>在二次抗体溶液中稀释1:100。 </td></tr><tr><td> Slowfade Gold抗淬灭试剂</td><td> Invitrogen公司的分子探针</td><td> S36936 </td><td>安装介质。储存在-20度。 </td></tr><tr><td> GlyceROL </td><td> Fisher Scientific则</td><td> G31-1 </td><td>对于安装。准备10毫升50％的稀释用蒸馏水，在室温下存储。 </td></tr><tr><td> CO 2 </td><td></td><td></td><td>对于麻醉成虫。 </td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td>两根锋利的镊子（杜蒙＃55） </td><td>精细科学工具</td><td> 11255-20 </td><td></td></tr><tr><td> Sylgard的184有机硅弹性体套件</td><td>道康宁</td><td></td><td>准备Sylgard的清扫用Sylgard混合物通过填充的塑料培养皿菜。 </td></tr><tr><td>三以及玻璃夹层菜</td><td> Fisher Scientific则</td><td> 21-379 </td><td></td></tr><tr><td>总重量Øminutien解剖针（直径0.1 mm）和两个pinholders（12厘米） </td><td>精细科学的工具</td><td> 26002-10 </td><td>插入一个minutien引脚在每个两pinholders和弯曲的标签中的一个，以形成一个钩子。 </td></tr><tr><td>显微镜盖玻片（22X22-1和24X40-1） </td><td> Fisher Scientific则</td><td> 12-542A </td><td></td></tr><tr><td>显微镜幻灯片（25x75x1.0毫米;预清洗） </td><td> Fisher Scientific则</td><td> 12-550-143 </td><td></td></tr><tr><td> 1.5 ml微量离心管</td><td></td><td></td><td></td></tr><tr><td>清除指甲油或透明胶带</td><td></td><td></td><td></td></tr><tr><td>轨道振动筛</td><td> Bellco </td><td></td><td></td></tr><tr><td>幻灯片水箱</tD&gt; <td> Fisher Scientific则</td><td></td><td></td></tr><tr><td>封口膜</td><td>比米斯</td><td> PM996 </td><td></td></tr><tr><td>薄画笔</td><td></td><td></td><td></td></tr><tr><td> P20和P200微量移液器和技巧</td><td></td><td></td><td></td></tr><tr><td>解剖显微镜</td><td></td><td></td><td></td></tr><tr><td>小水桶与冰</td><td></td><td></td><td>用于冷却的玻璃孔板，解剖视网膜和解决方案。 </td></tr></table>

dissection and immunohistochemistry of larval, pupal and adult drosophila retinas

果蝇的复眼由约750小眼（单位只眼）。每个小眼组成的约20个细胞，包括镜头的分泌视锥细胞，色素细胞，刷毛细胞和8感光体（的PR）R 1-R 8 2。永久居民有专门的微绒毛的结构，rhabdomeres，其中含有光敏感的的颜料，Rhodopsins（右轴）。 6的PR（R1-R6）的rhabdomeres形成一个梯形和包含Rh1对3 4。 R7和R8的rhabdomeres串联在梯形的中心被定位在共享相同的路径的光。 R7和R8永久居民的随机表达不同的组合RHS在两个主要的亚型5的&#39;p&#39;亚型，RH3 在 p R7S加上RH5 在 p R8S，而在&#39;Y&#39;亚型，与RH4 在 y R7S RH6 在 y，R8S 6 7 8。 PR和发展，就要有规范的早期开始在幼虫眼触角成虫盘，单层的上皮细胞。差异化的浪潮席卷盘9和启动组件的未分化细胞，成小眼10-11。的创始人细胞的R8被指定为第，并招募R1-6和R7 12-14。随后，在蛹的发展，PR分化导致广泛的形态学改变，15日 ，，包括rhabdomere形成，突触形成和最终RH表达。 在这个协议中，我们描述了在三个定义的视网膜发育期，可应用于视网膜的形成和发展途径，以解决各种问题的视网膜解剖和免疫组化的方法。在这里，我们使用这些的方法可视化逐步PR分化，在单细胞水平在整个安装幼虫，midpupal和成人视网膜（ <s豪华型&gt;图1）。

Watch this Scientific Journal Video about Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas at JoVE.com

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

“<em&gt;果蝇</em&gt;视网膜是一小部分的刻板的方式所产生的细胞类型组成的类似晶体的晶格<sup&gt; 1</sup&gt;。其顺从先进的遗传分析，使研究复杂的发展计划。本协议描述了视网膜的解剖和免疫组化在三个连续的发育阶段，与光感受器分化的重点。

幼虫，蛹和成人的解剖和免疫组化<em&gt;果蝇</em&gt;视网膜

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

dissection-immunohistochemistry-larval-pupal-adult-drosophila

Research

JoVE Journal

Neuroscience

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

25.1K 次观看.  New York University. “果蝇视网膜是一小部分的刻板的方式所产生的细胞类型组成的类似晶体的晶格 1。其顺从先进的遗传分析，使研究复杂的发展计划。本协议描述了视网膜的解剖和免疫组化在三个连续的发育阶段，与光感受器分化的重点。

视频: Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 expression is rarely tissue-specific enough to allow for precise correlation of the circuit with behavioral readouts. To overcome this major hurdle, we recently developed the FINGR method to achieve a more restrictive Gal4 expression in the tissue of interest. The FINGR method has three components: 1) the traditional Gal4/UAS system; 2) a set of FLP/FRT-mediated Gal80 converting tools; and 3) enhancer-trap FLP (ET-FLP). Gal4 is used to define the primary neural circuitry of interest. Paring the Gal4 with a UAS-effector, such as UAS-MJD78Q or UAS-Shits, regulates the neuronal activity, which is in turn manifested by alterations in the fly behavior. With an additional UAS-reporter such as UAS-GFP, the neural circuit involved in the specific behavior can be simultaneously mapped for morphological analysis. For Gal4 lines with broad expression, Gal4 expression can be restricted by using two complementary Gal80-converting tools: tubP&gt;Gal80&gt; ('flip out') and tubP&gt;stop&gt;Gal80 ('flip in'). Finally, investigators can turn Gal80 on or off, respectively, with the help of tissue-specific ET-FLP. In the flip-in mode, Gal80 will repress Gal4 expression wherever Gal4 and ET-FLP intersect. In the flip-out mode, Gal80 will relieve Gal4 repression in cells in which Gal4 and FLP overlap. Both approaches enable the restriction of the number of cells in the Gal4-defined circuitry, but in an inverse pattern. The FINGR method is compatible with the vast collection of Gal4 lines in the fly community and highly versatile for traditional clonal analysis and for neural circuit mapping. In this protocol, we demonstrate the mapping of FLP expression patterns in select ET-FLPx2 lines and the effectiveness of the FINGR method in photoreceptor cells. The principle of the FINGR method should also be applicable to other genetic model organisms in which Gal4/UAS, Gal80, and FLP/FRT are used.

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

We describe a Flippase-induced intersectional Gal80/Gal4 repression (FINGR) method, allowing tissue-specific FLP to determine Gal80 expression patterns. Wherever Gal4 and FLP overlap, Gal4 expression is turned on (Gal80 flipped out) or off (Gal80 flipped in). The FINGR method is versatile for clonal analysis and neural circuit mapping.

幼虫和成人测绘和增强子陷阱Flippase表达中的应用果蝇中枢神经系统

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 ...

科研

神经科学

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

单果蝇 Ommatidium解剖和影像

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

Dissection of Adult Mouse Utricle and Adenovirus-mediated Supporting-cell Infection

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear. Since there is no cell line that satisfactorily represents mammalian hair cells, research on hair cells relies on primary organ cultures. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice (Figure 1) 1-6. The utricle is a vestibular organ, and the hair cells of the utricle are similar in both structure and function to the hair cells in the auditory organ, the organ of Corti. The adult mouse utricle preparation represents a mature sensory epithelium for studies of the molecular signals that regulate the survival, homeostasis, and death of these cells.
Mammalian cochlear hair cells are terminally differentiated and are not regenerated when they are lost. In non-mammalian vertebrates, auditory or vestibular hair cell death is followed by robust regeneration which restores hearing and balance functions 7, 8. Hair cell regeneration is mediated by glia-like supporting cells, which contact the basolateral surfaces of hair cells in the sensory epithelium 9, 10. Supporting cells are also important mediators of hair cell survival and death 11. We have recently developed a technique for infection of supporting cells in cultured utricles using adenovirus. Using adenovirus type 5 (dE1/E3) to deliver a transgene containing GFP under the control of the CMV promoter, we find that adenovirus specifically and efficiently infects supporting cells. Supporting cell infection efficiency is approximately 25-50%, and hair cells are not infected (Figure 2). Importantly, we find that adenoviral infection of supporting cells does not result in toxicity to hair cells or supporting cells, as cell counts in Ad-GFP infected utricles are equivalent to those in non-infected utricles (Figure 3). Thus adenovirus-mediated gene expression in supporting cells of cultured utricles provides a powerful tool to study the roles of supporting cells as mediators of hair cell survival, death, and regeneration.

Mechanosensory hair cells are the receptor cells of the inner ear. The best-characterized in vitro model system of mature mammalian hair cells utilizes organ cultures of utricles from adult mice. We present the dissection of the adult mouse utricle, and we demonstrate adenovirus-mediated infection of supporting cells in cultured utricles.

解剖成年小鼠的椭圆囊和腺病毒介导的支撑细胞的感染

Hearing loss and balance disturbances are often caused by death of mechanosensory hair cells, which are the receptor cells of the inner ear.  Since there ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

在幼虫馈电电路的功能分析果蝇</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

的实时成像<em&gt;果蝇</em&gt;幼虫神经母细胞

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of the nervous system often produce viable larvae and adults that have locomotion defective phenotypes that are difficult to adequately describe with text or completely represent with a single photographic image. Current modes of scientific publishing, however, support the submission of digital video media as supplemental material to accompany a manuscript. Here we describe a simple and widely accessible microscopy technique for acquiring high-quality digital video of both Drosophila larval and adult phenotypes from a lateral perspective. Video of larval and adult locomotion from a side-view is advantageous because it allows the observation and analysis of subtle distinctions and variations in aberrant locomotive behaviors. We have successfully used the technique to visualize and quantify aberrant crawling behaviors in third instar larvae, in addition to adult mutant phenotypes and behaviors including grooming.

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Here we describe a simple and widely accessible microscopy technique to acquire high-quality digital video of Drosophila adult and larval mutant phenotypes from a lateral perspective.

收购高品质的数字视频<em&gt;果蝇</em&gt;从横向角度看幼虫及成虫行为

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

全山解剖和成年小鼠耳蜗的免疫

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

用线性琼脂糖渠道研究<em&gt;果蝇</em&gt;幼虫爬行行为

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

果蝇幼虫和成年脑的代谢分析

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...