Name: dissection and immunohistochemistry of larval, pupal and adult drosophila retinas
Uploaded: 2012-11-14T13:00:00
Description: Watch this Scientific Journal Video about Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas at JoVE.com

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?...

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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 ...

Research

JoVE Journal

Neuroscience

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

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

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

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

Single Drosophila Ommatidium Dissection and 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

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

Functional Analysis of the Larval Feeding Circuit in Drosophila

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

Live Imaging of Drosophila Larval Neuroblasts

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

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors 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

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

Using Linear Agarose Channels to Study Drosophila Larval 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

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

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

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

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 ...