We appreciate and would like to thank the Howard Hughes Medical Institute for the HHMI Professor award to U.B. which made this project possible. We thank the college at the University of California, Los Angeles for providing facilities and teaching infrastructure support for this work. The work was also supported with funding from Midwestern University and a generous donation from the Charity Fidelity Gift Fund. We thank John VandenBrooks for comments on the manuscript and Krista Pearman for her technical assistance.

这个过程是2天的过程。 第1天（2小时+解剖时） 1.蛹眼睛光盘解剖<ol><li>选择一个蛹解剖。 注:蛹被解剖的年龄将通过实验需要来确定。然而，如果检查细胞形态，这是经常做在puparium形成（APF）后42小时，在25℃，这是在视频中所示的蛹的年龄。收集白色蛹（视为0小时，APF）与润湿画笔并安排它们按时间顺序（基于收集时间）在保持于25℃的新飞食品小瓶中。解剖老龄蛹在此相同的时间顺序，以保持蛹尽可能接近之间的时间差异。 </li><li>转移蛹成一滴磷酸盐缓冲盐水（PBS，pH 7.4中，见材料配方）的聚硅氧烷解剖板。 </li><li>握住蛹壳钳和皮尔斯的顶部，并打开升蛹壳有一对锋利的钳的奥尔部分。从在新成立的孔的情况下取出蛹。 </li><li>做一个对角用剪刀剪，中胸部，在蛹。除去碎屑或蛹头转移到PBS的新下降。清洁用吸管鼓风机管（见材料和图1说明），并根据需要增加额外的PBS。一定要避免脱水。 </li><li>牢固地夹住蛹头角质层有一对镊子的顶端。使用吹风管的前端，轻轻推蛹壳挤出的脑和其他组织，包括眼的光盘，从蛹壳中。一定要避免角质层内抓住任何组织。 </li><li>使用吹风管充满PBS，轻轻吹脂肪人体组织和​​其他碎屑头的情况下清楚地分开，并确定了大脑和眼睛的光盘。 注:保持附着在蛹头壳体的大脑将允许研究者使用他广告的情况下，作为所述组织的未来操纵一个"把手"，而不会损坏眼睛光盘或脑。然而，这是常见的撞出从头部壳体的脑和眼睛的光盘，这是不利于整体过程和结果。往往需要吹和推成功地从内蛹头壳体和使它们清楚地从周围组织中分离提取出的眼盘和大脑的多次尝试。清洁器和多个分离的人能得到从周围组织中的眼睛的光盘将有助于防止外部组织从固定到眼睛的光盘。 </li><li>下面的夹层，立即将组织用0.5ml的修复溶液（见材料）转移到3以及玻璃皿置于冰上。浮在组织几分钟（通常花费解剖下一组眼盘的时间）的修复溶液的表面上。 注 :如果这不是第一眼盘在这一轮的清扫，completel被解剖Ÿ淹没以前的眼睛光盘进入修复解决方案。如果执行特殊染色程序（ 例如 ，活性氧，溶酶体等），这必须被固定之前完成。为了做到这一点，请将眼球解剖盘在冰冷的PBS之前，用适当的染色方案继续进行。 </li><li>以下夹层的所有眼光盘和浸没被解剖，将3以及玻璃盘到一个摇杆和岩石在室温下搅拌30分钟。 </li></ol> 2.免疫染色<ol><li>小心地取出修复溶液并加入0.5毫升的PBS与0.3％的Triton X-100（0.3％的PBT，见材料）和洗组织上的摇杆10分钟。总共4次洗涤（每次10分钟）重复此步骤3次以上。 注意:小心除去从孔中的溶液，可在解剖显微镜，以防止组织损伤或损失下进行。该溶液可以小心地用移液管吸出;我们更细tippeD，一次性移液管的已夷为平地，以减少组织吸的几率开幕。 </li><li>在第4次洗涤结束后，取出清洗和添加0.5毫升块溶液（见材料）到井和温育30分钟。 注:此时间段可以根据自己的需要进行扩展。 </li><li>在这30分钟块结束时，移液管加入10μl初级抗体溶液（稀释在方框溶液）放入井在微孔板的合适数量。 注:微孔板的一个井将容纳约4对附着在蛹壳眼睛光盘或8路隔离对眼睛光盘附带的大脑。在此协议为代表的数据所使用的一抗是抗 - 磷酸（1:500稀释）和抗剪切（1:200稀释）。 </li><li>转移的解剖组织成微孔板孔中填充了一抗溶液从3以及玻璃皿中。 </li><li>地方在润湿的纸巾的微孔板（防止脱水）在空吸管尖盒（或其他类似的容器），并储存在4℃下过夜。 </li></ol>第2天（4小时+的安装时间） <ol start="6"><li>传送从微孔板组织到一个新的3孔玻璃培养皿用0.3％的PBT（≈0.5毫升）洗所述组织上的摇杆10分钟。重复此步骤3次以上，总共4次洗涤（每次10分钟） </li><li>除去最后的洗涤和加入0.5毫升座溶液到孔中。阻断30分钟。 注:此时间段可以根据自己的需要进行扩展。 </li><li>从光盖或卸下铝块解决方案，并加入0.5毫升二抗溶液，以井（从适合你的一抗和荧光波长，在稀释液座荧光标记的抗体制成）和保护3以及玻璃盘贴膜孵育至少2小时，在室温温度。 注意:所有的后续步骤应同样不受光线照射。该温育可以扩展到过夜，在4℃下，以类似于第一抗体温育一个设置，但它也可以在覆盖着实验室膜，或​​者，如果抗体保护期望微孔板3以及玻璃盘进行。 </li><li>在抗体温育结束时，除去二抗溶液，并添加0.5毫升0.3％的PBT的孔，并且洗涤在摇臂10分钟。重复，共4次洗涤该步骤（每次10分钟）。 注:如果DAPI染色是期望的，在开始第一次洗涤之前，加入0.5微升的DAPI原液（见材料）与500微升的PBT，将用于对所述第一洗涤液（1:1000稀释）。 </li></ol> 3.安装<ol><li>在最后一次洗涤结束时，安装在滑动用70％的甘油或PBS触角/眼光盘作为介质以除去BR泉和在幻灯片上的一侧上的池其它不想要的组织。 </li><li>小心使用镊子之间的静水压力移动所述组织，或非常轻轻地用钳子前端，并在朝向滑动件的中心柱排队眼光盘。 </li><li>来自各地的组织去除所有多余的甘油或PBS（用实验室的毛细作用擦拭或镊子工作这一点）。 </li><li>放置安装介质的少量（≈10微升）沿着眼睛光盘柱的一侧和放置盖玻片上的组织，从而被散布在安装介质到眼睛上的光盘。 </li><li>让滑坐为1至2分钟，以允许组织/安装介质散开完全。 </li><li>来自各地的盖玻片的边缘擦拭任何额外的安装介质的切断，并用透明指甲油密封。 </li><li>捕捉利用共聚焦显微镜的眼睛光盘荧光图像。 </li></ol>

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The authors have nothing to disclose.

While it appears that the process is simple and easy to perform, in reality, this technique requires a great deal of practice to master. Routinely, we start students off by learning to dissect and mount third instar eye discs12, which are much easier to work with. This practice helps to develop an appropriate dissection position of the arms, hands and fingers13 so that manipulation of the forceps under the dissecting microscope is stable, easy and experienced. In essence, the practice period should achieve the goal of making the forceps into a natural extension of the fingers and thumb, allowing for the discrete and subtle movements required in the dissection. 
If using PBS to mount in, always have extra PBS available, as the small amount of PBS quickly evaporates in the mounting process. Another common problem is the folding of the eye discs in the Fix solution, thus fixing the eye disc in that orientation. This can be avoided by floating the eye disc on the surface of the Fix solution for some time (typically the length of the next eye disc dissection) before submerging it completely in the Fix solution.
While other published techniques focus on the dissection of the pupal eye disc at the midpupal stage9, when dissection is easily accomplished, this technique provides a method that can be applied to any pupal developmental time, except in the case of the pharate adult or early white pupa. We feel our technique provides an alternative and complementary approach to already published methods for pupal eye disc dissection. 
This technique, accompanied with common and useful genetic manipulations like FLP-FRT analysis14, UAS-GAL expression of transgenes15 and others, will allow researchers to study the eye disc in further detail during any pupal time frame.

果蝇 :发育和细胞生物学领域已经通过模式生物受到很大影响。在该模型中，眼睛光盘的研究已经作出了很大贡献有关知识信令，细胞生物学等领域。已故第三龄幼虫眼盘已被广泛研究，并是一个功能强大的模型利用，因为它给出了一系列发育阶段的快照，每个都有其自己的独特信号传导分子和过程，如在整个眼睛光盘的形态发生沟前进8。然而，有必要进一步扩大我们的发展过程的认识到发展的蛹阶段。虽然有研究蛹眼盘3-7，我们的知识不接近已经在三龄眼睛光盘已经完成的工作的广度。这是由于，在某种程度上，在解剖蛹眼睛光盘的更大的困难。因此，一个呈现的解剖适当的方法可以在这方面大大拓展研究。 虽然有在蛹眼睛光盘的发展阶段，很容易解剖，特别是围绕中期蛹期，其他时间段都更有挑战性解剖。该协议代表了一种方法解剖，可普遍用于所有蛹的发育时限蛹的眼睛光盘。这个协议可以用来作为一种替代另一种协议9，其示出了一种更简单和更快速的方法，从midpupal时间点解剖眼光盘。该协议最初拍摄，并在功能基因组学（URCFG）在蛹眼部解剖技术培训10,11先进的本科生在加州大学洛杉矶分校本科生研究协会开发的。许多本科学生能够利用这种视频和方法，了解这一具有挑战性的技术。

<table><tbody><tr><td>Phosphate-buffered saline (PBS, pH 7.4)</td><td></td><td></td><td>80 g NaCl, 2g&amp;nbsp; KCl, 14.4 g Na&lt;sub&gt;2&lt;/sub&gt;HPO4, 2.4 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, Bring volume to 1 &amp;#956;l, adjust the pH to 7.4, autoclave or filter sterilize, dilute to 1x PBS with autoclaved ddH&lt;sub&gt;2&lt;/sub&gt;O before using.</td></tr><tr><td>Triton X-100</td><td>Promega</td><td>H5142</td><td>Caution: Irritant! Wear gloves.</td></tr><tr><td>0.3% PBT</td><td></td><td></td><td>1.5 ml of Triton X-100, 500 ml 1X PBS.</td></tr><tr><td>37% formaldehyde solution</td><td>Fisher Scientific</td><td>F75P1GAL</td><td>Caution: Toxic, probable human carcinogen! Wear gloves.&amp;nbsp;</td></tr><tr><td>Fix Solution</td><td></td><td></td><td>(&amp;asymp;4% Formaldehyde in PBS) 50 &amp;mu;l of 37% Formaldehyde, 450 &amp;mu;l 1x PBS, make fresh before use</td></tr><tr><td>Normal goat serum</td><td>Rockland antibodies &amp;amp; assays</td><td>B304</td><td>Aliquot in 1 ml volumes and store at -80 &amp;#176;C</td></tr><tr><td>Block Solution</td><td></td><td></td><td>10% NGS in PBT.&amp;nbsp; This can be made and stored at 4 &amp;deg;C for a few days prior to use.</td></tr><tr><td>DAPI stock solution</td><td>Life Technologies</td><td>D3571</td><td>For coutnerstaining nuclei. Prepare a 1 mg/ml solution with ddH&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>VectaShield Mounting Medium</td><td>Vector Labs</td><td>H-1000</td><td>Mounting medium</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td>For mounting. Prepare 70% dilution with ddH&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Nutating mixer</td><td>VWR</td><td>82007-202</td><td>Used to rock tissue in 3 well glass dish</td></tr><tr><td>SylGard 182 Silicone Elastomer Kit</td><td>Krayden</td><td>NC9897184</td><td>Used to make silicone dissection dish</td></tr><tr><td>Silicone dissecting dish</td><td></td><td></td><td>Mix Sylgard elastomer kit (above) according to directions gently (to avoid bubbles). Pour mixture into Petri dish (any size). Allow SylGard to cure overnight in 37 &amp;deg;C incubator.</td></tr><tr><td>3 well glass dish</td><td>Corning</td><td>7220-85</td><td>The 3 well variety of these are no longer available, this is the 9 well product.</td></tr><tr><td>72 well microwell minitray</td><td>Nunc</td><td>438733</td><td></td></tr><tr><td>Sharp forceps (Dumont #55)</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>Vannas-type Micro Scissors, Straight, 5mm blade</td><td>Ted Pella</td><td>1346</td><td></td></tr><tr><td>100 mm Borosilicate glass capillaries</td><td>World Precision Instruments</td><td>1B100-4</td><td>Pull with needle puller to make fine point tip that allows a small stream of PBS to flow.</td></tr><tr><td>Disposable Transfer Pipets, Fine Tip</td><td>Samco Scientific</td><td>231</td><td></td></tr><tr><td>Tubing dimensions given are inner diameter (ID) x outer diameter (OD) x wall thickness in inches</td><td></td><td></td><td></td></tr><tr><td>PVC tubing (1/8 x 3/16 x 1/32)</td><td>Nalgene</td><td>8000-0010</td><td>Use these with pulled needle to assemble the blower tube as shown in Figure 2.</td></tr><tr><td>Tygon Silicone tubing (3/32 x 5/32 x 1/32)</td><td>Saint Gobain Performance Plastics</td><td>ABW00004</td><td></td></tr><tr><td>Tygon Silicone tubing (1/32 x 3/32 x 1/32)</td><td>Saint Gobain Performance Plastics</td><td>ABW00001</td><td></td></tr></tbody></table>

dissection and mounting of drosophila pupal eye discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

因为使用该协议的一个例子，结果示midpupal（42小时APF，在25℃）进行免疫染色用不同抗体眼光盘示于图2。通过使用针对磷酸酪氨酸残基的抗体，该细胞的膜可以是观察到的（ 图2A）。这可以被用来确定网膜细胞的规则排列在蛹眼以下之前midpupal阶段中发生的最终图案化工艺。另一个代表性图像描绘锥体细胞的细胞核可以通过使用抗体对剪切转录因子（ 图2B）来标识。 <img alt="图1" fo:content-width="4.5in" src="/files/ftp_upload/52315/52315fig1highres.jpg" width="450" /> 图1:在解剖过程中所用的送风机管的组装图对第细节参见材料节Ë独立的鼓风机管组件。 <img alt="图2" src="/files/ftp_upload/52315/52315fig2highres.jpg" /> 图2:中midpupal眼盘免疫染色染色代表演示如何使用该协议的可视化A）小眼（磷酸）和B）视锥细胞的细胞核（CUT）从蛹眼盘的顶形态在42小时APF。比例尺= 50微米。

Watch this Scientific Journal Video about Dissection and Mounting of Drosophila Pupal Eye Discs at JoVE.com

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

解剖和安装的<em&gt;果蝇</em&gt;蛹眼光盘

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

dissection-and-mounting-of-drosophila-pupal-eye-discs

Nanyang Technological University

Research

JoVE Journal

Biology

Dissection and Mounting of Drosophila Pupal Eye Discs

12.1K 次观看.  University of California at Los Angeles (UCLA). The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

视频: Dissection and Mounting of Drosophila Pupal Eye Discs

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

实时成像<em&gt;果蝇</em&gt;蛹眼

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

科研

发育生物学

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

从3龄果蝇幼虫的成虫光盘夹层

生物学

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

长期的实时成像<em&gt;果蝇</em&gt;眼睛光盘

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

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 ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

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 ...

神经科学

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

嗜酸性粒植物视网膜免疫组织化学、西方分析和 Rna 分离的解剖

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

一种新型的RFP的记者，以帮助在眼成虫光盘的可视化果蝇</em

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

成人的制备果蝇</em薄切片和显微镜分析>的眼睛

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

解剖和免疫染色成虫盘的<em&gt;果蝇</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

Dissection of the Eye-Brain Complex from Fly Pupae: A Method to Isolate Retinal Tissue

Source: DeAngelis, M. W., Johnson, R. I. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/59299/" target="_blank">Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation</a>. J. Vis. Exp.&#160;(2019).
This video describes how to dissect and isolate the eye-brain complex from Drosophila pupae. The featured protocol demonstrates the procedure yielding high-quality tissue, compatible with, for example, immunostaining, as well as, other gene expression experiments.

从苍蝇蛹中解剖眼脑复合物:一种分离视网膜组织的方法

Source: DeAngelis, M. W., Johnson, R. I. Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation. J. Vis. ...

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一种新型的RFP的记者，以帮助在眼成虫光盘的可视化果蝇

成人的制备果蝇的眼睛

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从苍蝇蛹中解剖眼脑复合物:一种分离视网膜组织的方法