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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Phosphate-buffered saline (PBS, pH 7.4)</td>
			<td></td>
			<td></td>
			<td>80g NaCl, 2g&#160; 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.</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.&#160;</td>
		</tr>
		<tr>
			<td>Fix Solution</td>
			<td></td>
			<td></td>
			<td>(&#8776;4% Formaldehyde in PBS) 50 &#956;l of 37% Formaldehyde, 450 &#956;l 1XPBS, make fresh before use</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Rockland antibodies &#38; assays</td>
			<td>B304</td>
			<td>Aliquot in 1 ml volumes and store at -80C</td>
		</tr>
		<tr>
			<td>Block Solution</td>
			<td></td>
			<td></td>
			<td>10% NGS in PBT.&#160; This can be made and stored at 4 &#176;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 ddH2O.</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 ddH2O.</td>
		</tr>
		<tr>
			<td colspan="4">Equipment</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 &#176;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><a name="RANGE!A19">Vannas-type Micro Scissors, Straight, 5mm blade</a></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 colspan="4">Tubing dimensions given are inner diameter (ID) x outer diameter (OD) x wall thickness in inches</td>
		</tr>
		<tr>
			<td>PVC tubing (1/8 x 3/16 x 1/32)</td>
			<td>Nalgene</td>
			<td>8000-0010</td>
			<td rowspan="3">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>
		</tr>
		<tr>
			<td>Tygon Silicone tubing (1/32 x 3/32 x 1/32)</td>
			<td>Saint Gobain Performance Plastics</td>
			<td>ABW00001</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

Research

JoVE Journal

Biology

Dissection and Mounting of Drosophila Pupal Eye Discs

12.0K 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

Dissection of Drosophila Ovaries

Drosophila 난소의 해부

연구

생물학

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Drosophila Melanogaster에서 애벌레 CNS의 해부

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

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

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

Drosophila 애벌레 NMJ의 해부

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

해부 그리고 스테 이닝 Drosophila 애벌레 난소

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

Drosophila Pupal의 복부 Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

의 번데기 케이스를 통해서 영상<em&gt; 초파리</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

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 ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

해부, 이미징 및 약물 학적 치료 : 초파리의 번데기 고환 및 단일 남성 생식 줄 낭종의 생체 내 문화

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology of the fish branchial skeleton is matched to a species' diet. Threespine stickleback fish (Gasterosteus aculeatus) have emerged as a model system to study the genetic and developmental basis of evolved differences in a variety of traits. Marine populations of sticklebacks have repeatedly colonized countless new freshwater lakes and creeks. Adaptation to the new diet in these freshwater environments likely underlies a series of craniofacial changes that have evolved repeatedly in independently derived freshwater populations. These include three major patterning changes to the branchial skeleton: reductions in the number and length of gill raker bones, increases in pharyngeal tooth number, and increased branchial bone lengths. Here we describe a detailed protocol to dissect and flat-mount the internal branchial skeleton in threespine stickleback fish. Dissection of the entire three-dimensional branchial skeleton and mounting it flat into a largely two-dimensional prep allows for the easy visualization and quantification of branchial skeleton morphology. This dissection method is inexpensive, fast, relatively easy, and applicable to a wide variety of fish species. In sticklebacks, this efficient method allows the quantification of skeletal morphology in genetic crosses to map genomic regions controlling craniofacial patterning.

The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species.

해부와 큰가시 고기 아가미 해골의 평면 장착

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology ...