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.

이 절차는 이일 절차이다. 1 일 (2 시간은 + 시간 해부) 1. 번데기 아이 디스크 해부 <ol><li> 해부의 번데기를 선택합니다. 참고 : 해부하는 번데기의 나이는 실험의 필요에 따라 결정됩니다. 세포 형태를 조사하는 경우에는,이 자주 비디오에 도시 번데기의 나이 인 25 ° C에서 puparium 형성 (APF) 후 42 시간에서 수행된다. 유체가 접촉되는 페인트 브러시와 흰색 번데기 (고려 0 시간의 APF)를 수집하고 25 ° C로 유지 새로운 플라이 식품 유리 병에 (수집 시간 기준) 시간 순서대로 배열합니다. 최대한 가까운 번데기 사이의 시간 차이를 유지하는 동일한 시간 순서로 세 번데기 해부. </li><li> 인산염 완충 식염수 한 방울에 번데기로 이동 실리콘 해부 접시에 (PBS, pH를 7.4, 조리법에 대한 자료를 참조). </li><li> 집게와 피어스와 번데기 케이스의 상단을 잡고 L을 엽니 다날카로운 집게 한 쌍의 번데기 케이스의 된 ower 부분. 새로 형성된 구멍의 경우에서 번데기를 제거합니다. </li><li> 번데기에서 가위, 중반 흉부와 대각선 컷을 확인합니다. 이물질을 제거 또는 PBS의 새로운 하락 번데기 머리를 전송합니다. 청소 피펫 송풍 튜브 (재료 및 자세한 내용은 그림 1 참조)를 사용하고 필요에 따라 추가 PBS를 추가합니다. 탈수를 방지해야합니다. </li><li> 안전하게 그립 집게 한 쌍의 번데기 머리 표피의 맨. 송풍 튜브의 끝 부분을 사용하여 부드럽게 뇌와 번데기 케이스의 밖으로 눈 디스크를 포함하여 다른 조직을 돌출 번데기 케이스에 밀어 넣습니다. 표피 내의 모든 조직을 잡아 않도록해야합니다. </li><li> PBS로 가득 송풍 튜브를 사용하여, 부드럽게 명확하게 분리하는 헤드 케이스에서 지방 신체 조직, 먼지 등을 날려 뇌와 눈 디스크를 식별합니다. 참고 : 번데기 헤드 케이스에 부착 된 뇌를 유지하는 것은 연구자가 자신을 사용하실 수 있습니다눈 디스크 나 뇌 손상을주지 않고 조직의 미래 조작에 대한 &quot;핸들&quot;로 광고하는 경우. 그러나, 헤드 케이스로부터 디스크 뇌와 눈을 제거하는 것이 일반적이며, 이것은 전체적인 절차 및 결과에 유해하지 않다. 그것은 종종 불고 성공적으로 그들이 분명히있는 주변 조직에서 분리하는 번데기 헤드 케이스 등 내에서 눈 디스크와 뇌를 추출하기 위해 추진하는 여러 시도를합니다. 깨끗하고 별도의 하나는 눈 디스크에 고정에서 외부 조직을 예방하는 데 도움이됩니다 주변 조직에서 눈 디스크를 얻을 수 있습니다. </li><li> 해부 이어 즉시 수정 용액 0.5 ㎖ (재료 참조) 얼음 3 웰 유리 접시에 조직을 전송. 몇 분 (이것은 눈 디스크의 다음 세트를 해부 걸리는 전형적 시간)에 대한 수정 용액의 표면 조직을 플로트. 참고 :이 해부, completel이 라운드에서 해부하는 최초의 아이 디스크가 아닌 경우Y는 수정 솔루션으로 이전 아이 디스크 잠수함. (등 예를 들어, 활성 산소 종, 리소좀,) 특수 염색 절차를 수행하면이 정착하기 전에 수행해야합니다. 이를 위해, 종래 적절한 염색 프로토콜로 계속 빙냉 PBS에서 해부 눈 디스크를 저장한다. </li><li> 모든 눈 디스크의 해부 및 침수를 수행하면 해부하고, 실온에서 30 분 동안 로커 바위에 3도 유리 접시를 이동합니다. </li></ol> 2. 면역 염색 <ol><li> 조심스럽게 수정 용액을 제거하고 0.3 % 트리톤 X-100과 0.5 ml의 PBS를 추가 (0.3 % PBT, 자료 참조)와 10 분 동안 로커에 조직을 씻어. 4 세척 (10 분 각각)의 단계를 총 세 번 이상 반복합니다. NOTE : 웰로부터의 용액을 조심스럽게 제거는 조직 손상 또는 손실을 방지하기 위해 해부 현미경 하에서 수행 될 수있다. 용액을 피펫으로 조심스럽게 흡인 될 수있다; 우리는 미세 tippe을 선호D, 조직의 흡인을 줄이려 평탄화 개구를 갖고 전사 일회용 피펫. </li><li> 넷째 세척이 끝나면, 세척을 제거하고 웰에 (재료 참조) 0.5 ml의 블록 용액을 추가 30 분 동안 배양한다. 참고 : 사용자의 필요에 따라 기간을 연장 할 수 있습니다. </li><li> 30 분의 시간 블록의 끝에서, 마이크로 웰 플레이트에서 웰의 적절한 수에 (블록 용액에 희석) 1 차 항체 용액 10 ㎕를 피펫. 주 : 마이크로 웰 플레이트 잘 하나가 번데기 케이스에 부착 된 눈 디스크의 약 4 쌍 또는 부착 두뇌와 눈 디스크의 8 격리 된 쌍을 수용 할 것입니다. (: 500 희석 (1)) 및 항 - 컷 (1 : 200 희석) 대표 데이터는이 프로토콜에서 사용 된 주요 항체는 항 - 포스이다. </li><li> 3 잘 유리 접시에서 일차 항체 용액으로 충전 마이크로 웰 플레이트의 우물에 해부 조직을 전송합니다. </li><li> 장소젖은 종이 타월에 마이크로 웰 플레이트는 4 ° C에서 하룻밤 빈 피펫 팁 상자 (또는 기타 유사한 용기) 및 저장소 (탈수를 방지하기 위해). </li></ol> 2 일 (4 시간 + 시간 장착) <ol start="6"><li> 0.3 % PBT (≈0.5 ml)로 새로운 3 웰 유리 접시에 마이크로 웰 플레이트에서 조직을 전송하고 로커에 10 분 동안 조직을 씻는다. (4)의 세척 단계를 총 (10 분마다)을 세 번 이상 반복 </li><li> 마지막 세척을 제거하고 잘에 차단 솔루션의 0.5 ml를 추가합니다. 30 분 동안 차단합니다. 참고 : 사용자의 필요에 따라 기간을 연장 할 수 있습니다. </li><li> 덮개 또는 알루미늄의 빛에서 3 웰 유리 접시를 차단 솔루션을 제거하고 잘에 (블록 용액에 희석 기본 항체와 형광 파장에 적합한 형광 표지 항체에서 만든) 차 항체 용액 0.5 ml의 추가 및 보호 박 및 방에서 적어도 2 시간 동안 배양온도. 주 : 이후의 모든 단계가 유사하게 빛 노출로부터 보호되어야한다. 이 배양 차 항체 인큐베이션 유사한 설정에서, 4 ° C에서 밤새 확장 될 수 있지만, 또한 실험실 막 또는 항체 절약이 요구되는 경우에는 마이크로 웰 플레이트로 덮여 3 웰 유리 접시에 수행 될 수있다. </li><li> 항체 인큐베이션의 끝에, 차 항체 용액을 제거하고 웰에 0.3 % PBT 0.5를 가하여 10 분간 로커에 씻는다. 4 세척의 총에 대해이 단계 (10 분마다)를 반복합니다. NOTE : (: 1000 희석 한) DAPI 염색이 필요한 경우, 첫 번째 세척을 시작하기 전에, 처음 세탁에 사용되는 PBT의 500 μL에 DAPI 저장 용액 0.5 μl를 (자료를 참조)에 추가. </li></ol> 3. 장착 <ol><li> 마지막 세척이 끝나면, BR을 제거하는 매체로서 70 % 글리세롤 또는 PBS를 사용하여 슬라이드 더듬이 / 눈 디스크 마운트아인과 슬라이드의 일측에 풀 기타 원치 않는 조직. </li><li> 조심스럽게 집게 사이의 수압을 이용하여 조직을 이동하거나 아주 부드럽게 포셉 팁, 및 상기 슬라이드의 중심을 향해 열에 눈 디스크를 일렬로. </li><li> 조직 주위에서 모든 외부 글리세롤 또는 PBS를 제거 (실험실의 위킹 조치를 닦아 사용하거나 집게이 작동). </li><li> 눈 디스크 열의 일측을 따라 장착 매체의 소량 (≈10 μL)을 놓고 눈 디스크에 장착 매질을 전파하도록 조직에 커버 슬립을 배치. </li><li> 1 ~ 2 분은 조직 / 설치 매체가 완전히 확산 할 수 있도록하는 슬라이드 쉬십시오. </li><li> 커버 슬립의 가장자리에서 모든 추가 설치 매체를 닦아 맑은 매니큐어로 밀봉. </li><li> 공 초점 현미경을 이용하여 안구 디스크의 형광 이미지를 캡처. </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 shou...

초파리 melanogaster : 개발 및 세포 생물학의 분야는 크게 모델 생물에 의해 영향을하고있다. 이 모델 내에서 눈 디스크의 연구는 지식에 관한 신호 전달, 세포 생물학 및 다른 분야의 큰 거래를 기여했다. 후반 세 번째 애벌레 령 눈 디스크가 광범위하게 연구하고 개발 기간의 일련의 스냅 샷을 제공합니다으로 활용하는 강력한 모델 된, 고유의 신호 분자 및 프로세스 각각은, 형태 형성 고랑은 눈 디스크에 걸쳐 진행 8. 그러나, 앞으로의 개발 번데기 단계로 발달 과정에 대한 이해를 확장 할 필요가있다. 번데기 눈 디스크 3-7에 연구가되었지만, 우리의 지식은 세 번째 령 눈 디스크에 수행 된 작업의 폭을 접근하지 않습니다. 이 번데기 눈 디스크를 해부에 큰 어려움으로 부분적 때문이다. 따라서, 프레 젠 테이션해부의 적절한 방법은 크게이 지역에서 연구를 확장 할 수있다. 쉽게 특히 중반 번데기 기간의 주위에, 해부 번데기 눈 디스크 개발 내에서 단계가 있지만, 다른 기간은 훨씬 더 도전적인 해부한다. 이 프로토콜은 보편적으로 모든 번데기 발달 타임 프레임에 사용될 수 번데기 눈 디스크를 해부위한 하나의 방법을 나타낸다. 이 프로토콜은 midpupal 시점에서 눈 디스크를 해부 쉽고 빠른 방법을 나타낸 다른 프로토콜 (9)의 대안으로 사용될 수있다. 이 프로토콜은 원래 촬영 및 기능 유전체학 (URCFG) 번데기 눈 해부의 기술에서 10, 11에서 UCLA 학부 연구 컨소시엄에서 고급 학부 학생들을 훈련을 위해 개발되었다. 많은 학부 학생들이 도전적인 기술을 알아 보려면이 비디오 및 방법을 활용할 수 있었다.

<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.

이 프로토콜의 사용의 예로서, 결과 midpupal (42 시간 동안 25 ° C에서 APF) 상이한 항체로 면역 염색 눈 디스크를 나타내는 주 포스 포 티로신 잔기에 대해 유도 된 항체를 사용하여도 2에 제시되어, 세포의 멤브레인 일 수있다 관찰 (그림 2A). 이것은 midpupal 단계 이전에 발생하는 최종 패터닝 공정 다음 번데기 눈 ommatidial 세포의 정규 배열을 식별하는 데 사용될 수있다. 또 다른 대표 화상?...

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.

해부와의 설치<em&gt; 초파리</em&gt; 번데기 아이 디스크

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 de Cartagena UDEC

Research

JoVE Journal

Biology

Dissection and Mounting of Drosophila Pupal Eye Discs

12.1K 조회수.  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.

비디오: 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.

의 라이브 영상<em&gt; 초파리</em&gt; 번데기 눈

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

연구

발생학

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.

셋째 Instar Drosophila의 유충에서 Imaginal 디스크의 해부

생물학

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.

장기의 라이브 영상<em&gt; 초파리</em&gt; 아이 디스크

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.

Drosophila 아이 Imaginal 디스크에 Glial 세포 이미징 마이 그 레이션 라이브

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.

애벌레의, Pupal 및 성인의 해부 및 Immunohistochemistry<em&gt; Drosophila</em&gt; 망막

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

신경과학

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;

Immunohistochemistry, 서쪽 분석 및 RNA 격리에 대 한 초파리 번데기 망막의 해 부

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.

에있는 아이 Imaginal 디스크의 시각화에 도움을 소설 RFP 리포터 Drosophila</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.

성인의 준비 Drosophila</em씬 Sectioning 및 현미경 분석을위한> 눈

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.

해부하고, 상상 디스크의 면역 염색에서<em&gt; 초파리</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://jove.unicartagenaproxy.elogim.com/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.

파리 번데기에서 눈-뇌 복합체의 해부: 망막 조직을 분리하는 방법

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

해부와의 설치

요약

더 많은 비디오 탐색

의 라이브 영상

셋째 Instar Drosophila의 유충에서 Imaginal 디스크의 해부

장기의 라이브 영상

Drosophila 아이 Imaginal 디스크에 Glial 세포 이미징 마이 그 레이션 라이브

애벌레의, Pupal 및 성인의 해부 및 Immunohistochemistry

Immunohistochemistry, 서쪽 분석 및 RNA 격리에 대 한 초파리 번데기 망막의 해 부

에있는 아이 Imaginal 디스크의 시각화에 도움을 소설 RFP 리포터 Drosophila

성인의 준비 Drosophila 눈

해부하고, 상상 디스크의 면역 염색에서

파리 번데기에서 눈-뇌 복합체의 해부: 망막 조직을 분리하는 방법