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.

この手順では、2日間の処置である。 1日目（2時間+解剖時間） 1.蛹アイディスク解剖<ol><li>解剖用の蛹を選択します。 注：解剖する蛹の年齢は実験的なニーズによって決定されます。しかし、細胞の形態を検査した場合、これはしばしば、ビデオに示す蛹の年齢である25℃、蛹殻形成（APF）の後42時間で行われる。濡れた絵筆で（0時間APF考えられる）白い蛹を収集し、25℃に維持し、新しいフライ食品バイアル中で（収集時間に基づいて）時系列順に並べ。できるだけ近い蛹間の時間差を維持するために、この同じ時系列順に熟成蛹を解剖。 </li><li>シリコーン解剖板上のリン酸緩衝生理食塩水（PBS、pH7.4で、レシピの材料を参照のこと）の低下に蛹を転送する。 </li><li>鉗子やピアスと蛹のケースの上部を持ち、Lを開く鋭いピンセットで蛹ケースのower部分。新たに形成された穴にケースから蛹を削除します。 </li><li>蛹でハサミで斜めカット、半ば胸部を作る。破片を除去またはPBSの新しいドロップに蛹ヘッドを移す。ピペット送風管を使用してクリーニングし（詳細は材料および図1を参照）、必要に応じて追加のPBSを加える。脱水症状を避けるために確認してください。 </li><li>しっかりとグリップピンセットで蛹頭部キューティクルの最上部。送風管の先端を使用して、穏やかに蛹のケースから、眼のディスクを含む、脳および他の組織を押し出すために蛹のケースに押し込み。キューティクル内の任意の組織をつかん避けるようにしてください。 </li><li> PBSを充填した送風管を使用して、穏やかに明確に分離し、脳および目のディスクを識別するために、ヘッドケースから脂肪体組織および他の破片を吹く。 注：蛹ヘッドケースに付着した脳を維持することは、研究者が彼を使用できるようになります目のディスクや脳に損傷を与えることなく、組織の将来の操作のための「ハンドル」として広告のケース。しかし、ヘッドケースから脳および目のディスクを除去することが一般的であり、これは全体的な手順および結果に有害ではない。それは頻繁に成功蛹ヘッドケース内および彼らが周囲の組織から明確に分離されるように、目からディスクと脳を抽出するために吹いてプッシュする複数の試みを取る。よりクリーンで別々の1は、目のディスクに固定から余分な組織を防ぐことができます周囲の組織からのアイディスクを得ることができます。 </li><li>切開の後、すぐに修正溶液0.5ml（材料を参照）、氷上で3ウェルガラス皿に組織を移す。数分間定着液（それは目のディスクの次のセットを分析するのにかかる時間は、通常）の表面上の組織フロート。 注記 ：これは解剖のこのラウンドで解剖された最初のアイディスクでない場合、completelyは修正液中に前のアイディスクを水没。特殊な染色手順（ 例えば 、活性酸素種、リソソームなど）を実行する場合、これは固定前に行う必要があります。これを行うために、適切な染色プロトコルを継続する前に、氷冷PBS中で解剖目ディスクを格納する。 </li><li>解剖されているすべてのアイディスクの解剖や水没に続いて、30分間室温でロッカーや岩に3ウェルガラス皿を移動します。 </li></ol> 2.免疫染色<ol><li>慎重に修正液を除去し、0.3％トリトンX-100を含むPBSを0.5mlを加える（0.3％PBT、材料を参照）、10分間、ロッカー上で組織を洗浄する。 4回の洗浄（各10分）の合計のために、この手順を3回繰り返します。 注意：ウェルから溶液を注意深く除去は、組織の損傷または損失を防止するために、解剖顕微鏡下で行うことができる。溶液はピペットで慎重に吸引することができる。我々は、ファインtippeを好むdは、開口部は、組織を吸引する可能性を低減するために平坦化されてきた使い捨てトランスファーピペット。 </li><li>第4回の洗浄の最後に、洗浄液を除去し、ウェルに（材料を参照）0.5ミリリットルブロック溶液を追加し、30分間インキュベートする。 注：この時間枠は、あなたのニーズに応じて拡張することができます。 </li><li> 30分間のブロック時間の終了時に、マイクロウェルプレートのウェルに適切な数にする（ブロック溶液中に希釈された）一次抗体溶液の10μlをピペット。 NOTE：マイクロウェルプレートの一つのウェルは蛹ケースに取り付けアイディスクの約4対または添付脳眼ディスクの8孤立対を収容する。 （：500希釈1）および抗カット（1：200希釈）代表的なデータについては、このプロトコルで使用される一次抗体は、抗ホスホチロシンです。 </li><li> 3ウェルガラス皿から一次抗体溶液を充填したマイクロウェルプレートのウェルに解剖した組織を移す。 </li><li>場所湿らせたペーパータオルの上マイクロウェルプレートは、4℃で一晩、空のピペットチップボックス（または他の類似の容器）とストア内（脱水を防ぐため）。 </li></ol> 2日目（4時間+実装時間） <ol start="6"><li> 0.3％PBT（≈0.5ml）で新しい3ウェルガラス皿にマイクロウェルプレートからの組織を移し、ロッカー上で10分間組織を洗う。 4回の洗浄（各10分）の合計のために、このステップを3回以上繰り返します</li><li>最後の洗浄液を取り出し、ウェルにブロック溶液0.5mlを加える。 30分間ブロックする。 注：この時間枠は、あなたのニーズに応じて拡張することができます。 </li><li>ブロック溶液を除去し、ウェルに二次抗体溶液（ブロック溶液中に希釈し、あなたの一次抗体および蛍光波長に適した蛍光標識された抗体から作られる）の0.5ミリリットルを追加し、カバーまたはアルミニウムで光から3ウェルガラス皿を保護箔、室温で少なくとも2時間インキュベート温度。 注：すべてのその後の工程は、同様に、露光から保護されるべきである。このインキュベーションは、一次抗体のインキュベーションと同様のセットアップで、4℃で一晩延長することができるが、抗体の保存が望まれる場合、それはまた、実験用フィルム、またはマイクロウェルプレートで覆われた3ウェルガラス皿中で行うことができる。 </li><li>抗体インキュベーションの終わりに、二次抗体溶液を除去し、ウェルに0.3％PBT 0.5mlを追加し、10分間ロッカー上洗う。 4回の洗浄（各10分）の合計に対して、この手順を繰り返します。 注：（：1,000希釈1）DAPI染色が必要な場合は、最初の洗浄を開始する前に、最初の洗浄のために使用されるPBTの500μlに（資料を参照）、DAPIストック溶液0.5μlを添加する。 </li></ol> 3.取付<ol><li>最後の洗浄の終わりには、brのを除去するための媒体としての70％グリセロールまたはPBSを使用して、スライド上の触角/眼、ディスクをマウントアインスライドの一方の側に、プール内の他の望ましくない組織。 </li><li>慎重に鉗子間の静水圧を用いて組織を動かし、または非常に優しく鉗子先端で、スライドの中心に向かって列内の目のディスクをラインアップ。 </li><li>組織の周りからすべての余分なグリセロールまたはPBSを取り外します（研究室のウィッキング作用を利用してワイプまたは鉗子は、このために働く）。 </li><li>目のディスク列の一辺に沿って封入剤を少量（≈10μl）を置き、目のディスクに封入剤を広がるように組織上カバースリップを置く。 </li><li>スライドが完全に広がる組織/封入を可能にするために1〜2分間放置します。 </li><li>カバースリップのエッジの周りから余分な封入剤を拭き取り、明確なマニキュアでそれを封印。 </li><li>共焦点顕微鏡を用いて眼ディスクの蛍光画像をキャプチャします。 </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.

キイロショウジョウバエ ：発達と細胞生物学の分野が大幅にモデル生物の影響を受けてきた。このモデルの中では、目のディスクの研究はシグナル伝達、細胞生物学やその他の分野に関する知識を大いに貢献してきました。後半に第三の幼虫齢目のディスクが広く研究し、それが発達期間の一連のスナップショットを与えるように、利用するための強力なモデルであるされている、独自のシグナリング分子、およびプロセスとのそれぞれが、形態形成溝は、目のディスクを横切って進むにつれて8。しかし、更なる発展の蛹の段階に発達過程の理解を拡大する必要性がある。蛹のアイディスク3-7の研究がなされてきた一方で、私たちの知識は3齢目のディスクに行われている仕事の幅広さに近づかない。これは、蛹のアイディスク解剖で大きな困難に一部起因している。したがって、プレゼンテーション解剖の適切な方法が大幅にこの分野の研究を展開することができます。 簡単に、特に中旬蛹期間を中心に、解剖された蛹のアイディスク開発中の段階がありますが、他の期間は、はるかに困難な解剖べきである。このプロトコルは、普遍的にすべての蛹の発育時間枠のために使用することができる蛹目ディスクを切開するための1つの方法を表している。このプロトコルは、midpupal時点から目ディスクを分析するための簡単かつ迅速に方法を示す別のプロトコル9の代替として使用することができる。このプロトコルは、もともと撮影さと機能ゲノミクス（URCFG）蛹眼の解剖の手法で10,11でUCLAの学部研究コンソーシアムで高度な学部学生を訓練するために開発されました。多くの学部学生がこのような厳しいテクニックを学ぶためにこのビデオと方法を利用することができました。

<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.

このプロトコルの使用例として、異なる抗体で免疫染色midpupalを示す結果（42時間APF 25°Cで）眼のディスクはホスホチロシン残基に対する抗体を使用することにより、図2に示されている、細胞の膜とすることができる観測された（ 図2A）。これはmidpupal段階の前に発生する最後のパターニングプロセス以下蛹眼の個眼細胞の規則的な配列を同定するために用いることができる。別の代表的な画像は、錐体細胞の核がカット転写因子（ 図2B）に対する抗体を用いて同定されることができ示す。 <img alt="図1" fo:content-width="4.5in" src="/files/ftp_upload/52315/52315fig1highres.jpg" width="450" /> 図1：解剖手順で使用送風管のアセンブリー·ダイアグラム番目の詳細については、材料のセクションを参照してください送風管のeは、個別のコンポーネント。 <img alt="図2" src="/files/ftp_upload/52315/52315fig2highres.jpg" /> 図2：midpupal目のディスクの免疫染色代表免疫染色は、Aを視覚化するためにこのプロトコルの使用を実証する）個眼の頂端形態（ホスホチロシン）とB）コーン細胞核（カット）42時間APFでの蛹のアイディスクから。スケールバー=50μmである。

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.

解剖と実装<em&gt;ショウジョウバエ</em&gt;蛹アイディスク

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

Universidad San Sebastian

Research

JoVE Journal

Biology

Dissection and Mounting of Drosophila Pupal Eye Discs

12.1K ビュー.  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.

ビデオ: 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.

のライブイメージング<em&gt;ショウジョウバエ</em&gt;蛹アイ

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

発生生物学

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.

3齢幼虫ショウジョウバエの幼虫から成虫ディスクの解剖

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.

長期のライブイメージング<em&gt;ショウジョウバエ</em&gt;アイディスク

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.

ショウジョウバエの眼成虫ディスクに付属してグリア細胞の移行のイメージングを生きる

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、蛹および成人の解剖と免疫組織化学<em&gt;ショウジョウバエ</em&gt;網膜

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

神経科学

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;

免疫組織化学、西部の分析、および RNA の隔離のためのショウジョウバエ蛹網膜の解剖

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.

のアイ成虫原基の可視化を支援するために新しいRFPレポーターショウジョウバエ</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.

大人の準備ショウジョウバエ</emシンセクショニングおよび顕微鏡分析のための>アイズ

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.

解剖や成虫の免疫染色から<em&gt;キイロショウジョウバエ</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.bdigitaluss.remotexs.co/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.

ハエの蛹からの眼脳複合体の解剖:網膜組織を分離する方法

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

解剖と実装

要約

さらに動画を探す

のライブイメージング

3齢幼虫ショウジョウバエの幼虫から成虫ディスクの解剖

長期のライブイメージング

ショウジョウバエの眼成虫ディスクに付属してグリア細胞の移行のイメージングを生きる

幼虫、蛹および成人の解剖と免疫組織化学

免疫組織化学、西部の分析、および RNA の隔離のためのショウジョウバエ蛹網膜の解剖

のアイ成虫原基の可視化を支援するために新しいRFPレポーターショウジョウバエ

大人の準備ショウジョウバエアイズ

解剖や成虫の免疫染色から

ハエの蛹からの眼脳複合体の解剖:網膜組織を分離する方法