해부와의 설치<em> 초파리</em> 번데기 아이 디스크
Summary
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.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Phosphate-buffered saline (PBS, pH 7.4) | 80g NaCl, 2g KCl, 14.4g Na2HPO4, 2.4g KH2PO4, Bring volume to 1 l, adjust the pH to 7.4, autoclave or filter sterilize, dilute to 1X PBS with autoclaved ddH2O before using. | ||
| Triton X-100 | Promega | H5142 | Caution: Irritant! Wear gloves. |
| 0.3% PBT | 1.5 ml of Triton X-100, 500 ml 1X PBS. | ||
| 37% formaldehyde solution | Fisher Scientific | F75P1GAL | Caution: Toxic, probable human carcinogen! Wear gloves. |
| Fix Solution | (≈4% Formaldehyde in PBS) 50 μl of 37% Formaldehyde, 450 μl 1XPBS, make fresh before use | ||
| Normal goat serum | Rockland antibodies & assays | B304 | Aliquot in 1 ml volumes and store at -80C |
| Block Solution | 10% NGS in PBT. This can be made and stored at 4 °C for a few days prior to use. | ||
| DAPI stock solution | Life Technologies | D3571 | For coutnerstaining nuclei. Prepare a 1 mg/ml solution with ddH2O. |
| VectaShield Mounting Medium | Vector Labs | H-1000 | Mounting medium |
| Glycerol | Sigma | G5516 | For mounting. Prepare 70% dilution with ddH2O. |
| Equipment | |||
| Nutating mixer | VWR | 82007-202 | Used to rock tissue in 3 well glass dish |
| SylGard 182 Silicone Elastomer Kit | Krayden | NC9897184 | Used to make silicone dissection dish |
| Silicone dissecting dish | 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 °C incubator. | ||
| 3 well glass dish | Corning | 7220-85 | The 3 well variety of these are no longer available, this is the 9 well product. |
| 72 well microwell minitray | Nunc | 438733 | |
| Sharp forceps (Dumont #55) | Fine Science Tools | 11255-20 | |
| Vannas-type Micro Scissors, Straight, 5mm blade | Ted Pella | 1346 | |
| 100 mm Borosilicate glass capillaries | World Precision Instruments | 1B100-4 | Pull with needle puller to make fine point tip that allows a small stream of PBS to flow. |
| Disposable Transfer Pipets, Fine Tip | Samco Scientific | 231 | |
| Tubing dimensions given are inner diameter (ID) x outer diameter (OD) x wall thickness in inches | |||
| PVC tubing (1/8 x 3/16 x 1/32) | Nalgene | 8000-0010 | Use these with pulled needle to assemble the blower tube as shown in Figure 2. |
| Tygon Silicone tubing (3/32 x 5/32 x 1/32) | Saint Gobain Performance Plastics | ABW00004 | |
| Tygon Silicone tubing (1/32 x 3/32 x 1/32) | Saint Gobain Performance Plastics | ABW00001 | |
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