우리는 P. Verrijzer, J. 요한센 H., 항체에 대한 오쿠라와 A. Debec, 블루밍턴과 비엔나 주식 센터에 파리에 감사드립니다. 제안과 자극 토론에 대한 C. 잭슨 감사합니다.

1. 드로소필라 고환 컬렉션
참고: 젊은 남성 (0-15 h 포스트 백과) diploid 세균 세포를 검사하는 데 필요합니다 (즉, 정자 고니아 및 정자 세포).
<ol><li>정자 개발에서 간섭을 최소화하기 위해 가능한 한 많이 젊은 파리를 선택합니다.</li>
	<li>이산화탄소에 잠깐 노출되어 파리를 마취하고 플라이 패드14,15로옮김한다.</li>
	<li>해부 현미경 (10 배율)에서, 머리를 제거하기 위해 집게를 사용합니다.</li>
	<li>5~10개의 참수된 파리를 링거의 완충제(183m MM KCI, 47m NaCl, 10mM Tris-HCI, 1mM EDTA, 완전한 단백질 억제제, pH 6.8)로 이동하여 검은 배경 지지대에서 실리콘 코팅 유리 슬라이드를 장착한다.</li>
	<li>40배율에서 해부현미경으로, 흉부와 복부 사이를 날아다니는 5번 집게 1쌍을 사용하고, 다른 집게와 함께 흉부에서 복부를 끌어당깁니다. 고환과 인접한 조직을 플라이시체(15)에서 빠르게 격리하고 링거의 버퍼를 같은 슬라이드에 새로 한 방울 로 떨어뜨립니다.</li>
	<li>모든 고환이 준비되면 나머지 조직 (액세서리 선 및 외부 생식기)에서 고환을 청소하십시오.</li>
	<li>해부된 고환을 포함하는 낙하에서 초과 버퍼를 제거합니다. 일반적으로 200 μL 팁 위에 10 μL 팁이 장착된 p200 파이펫을 사용하십시오. 이렇게 하면 작은 조리개를 통해 액체 과잉을 제거할 수 있습니다. 해부 된 고환을 포함하는 드롭 위에 4 %의 파라 포름알데히드 고정을 한 다음 고환을 신선한 고정 솔루션을 포함하는 튜브로 신속하게 전송합니다.</li>
</ol>2. 파라포름알데히드 고정
<ol><li>사용하기 직전에 2mL의 고정 용액을 준비하십시오: NP40 20%의 30 μL과 2m 실리콘 마이크로센트리슈지 튜브에 1200 μL의 30μL을 가진 인산염 완충식식염(PBS)에서 희석된 4% 파라포름알데히드의 600 μL. 600 μL 알리쿼트에 제조및 -20 °C에 저장 PBS에서 4 %의 파라 포름 알데히드를 사용합니다. 주의: 헵탄과 파라포름알데히드는 독성이 있으며 연기 후드로 처리해야 합니다. 호스트 연구소의 규정에 따라 처분하십시오.</li>
	<li>PFA/NP40/heptane 용액에 넓은 조리개 팁이 있는 p200 마이크로파이펫을 사용하여 고환을 전송합니다. 튜브를 30s의 위아래로 흔들어 줍니다. 실온에서 로터리 믹서에 고환을 30분 동안 계속 고정하십시오.</li>
	<li>로터리 믹서를 중지하고 튜브랙에 튜브를 배치하여 위상 분리를 허용하고 고환이 튜브의 바닥에 정착할 수 있도록 합니다.</li>
	<li>모든 heptane 및 고정 솔루션을 제거하고 1x PBS로 고환을 세 번 빠르게 헹구습니다.</li>
	<li>깨끗한 200 μL 개별 튜브로 옮김합니다. 이 작은 관에 있는 모든 잠복기를 수행하여 채택된 항체의 양을 최소화하십시오.</li>
	<li>P200과 P10 팁이 모두 부착된 200 μL 마이크로파이프를 사용하여 초과 버퍼를 제거합니다. 고환을 흡인하지 않도록 주의하십시오.</li>
	<li>모든 샘플이 준비될 때까지 고환을 얼음 위에 1배 PBS로 보관하십시오.</li>
</ol>3. 용액에 항체 염색
<ol><li>PBS를 폐기하고 세포막을 투과화하기 위해 실온에서 30 분 동안 0.3 % PBS-T로 고환을 둡니다.</li>
	<li>동일한 버퍼에 사전 배양하고 1h의 일반 염소 혈청(NGS)을 5% 더한 값입니다.</li>
	<li>0.3% PBS-T 플러스 5% NGS(재료표)로 희석된 1차 항체의 200 μL을추가합니다.</li>
	<li>로커에 부드러운 동요와 함께 4 °C (또는 실온)에서 하룻밤 기본 항체를 배양.</li>
	<li>실온에서 로커에서 15 분 동안 0.3 % PBS-T에서 3 번 세척하십시오. 고환이 중력에 의해 튜브의 바닥에 정착할 수 있도록 합니다.</li>
	<li>DyLight 공액 계열에서 희석된 이차 항체 의 200 μL을 1:500로 추가합니다.</li>
	<li>실온에서 4 시간 동안 어두운 챔버에서 이차 항체를 배양하십시오.</li>
	<li>실온에서 로커에서 0.3% PBS-T로 15분 간 세척합니다. 그런 다음 0.2 % PBS-T와 0.1 % PBS-T로 반복되며 실온에서 로커에서 30 분 동안 매번 반복하십시오. 세제를 제거하기 위해 30 분 동안 1 x PBS에서 마지막 세척을하십시오.</li>
	<li>1x PBS에 1μg/mL에 180 μL의 DAPI 용액을 15분 간 추가합니다. 주식 용액은 H2O. PBS에서 재고 용액을 만들지 않도록 10 mg / mL이며, 이는 더 큰 정자 세포에서 확산 크로 마틴에 DAPI 염색 신호를 줄일 수 있습니다.</li>
	<li>각각 3배씩 5분 동안 씻으시다.</li>
	<li>소수성 장벽 펜을 사용하여 깨끗한 슬라이드에 원을 그립니다.</li>
	<li>흡습 고환을 넣고 슬라이드에 넣습니다. 0.1% PBS-T로 넓은 조리개 팁을 미리 헹구면 고환이 팁에 달라붙지 않도록 합니다. 초과 PBS를 제거합니다.</li>
	<li>Citifluor AF1의 드롭(약 100 μL)을 추가합니다.</li>
	<li>유리 덮개 를 조직 위에 부드럽게 놓고 기포를 포획하지 않도록주의하십시오.</li>
	<li>투명 매니큐어를 사용하여 커버슬립을 슬라이드에 밀봉합니다.</li>
	<li>현미경에 슬라이드를 관찰.</li>
</ol>4. 지역화 분석
<ol><li>공초점 이미지를 수집합니다. 우리는 0.5 또는 1 μm의 z 해상도와 자이스 LSM 710 공초점 현미경 (63x 계획 아포크로틱 오일)을 사용했다.</li>
	<li>독립적 인 고환에서 다른 세포를 선택합니다.</li>
	<li>이미지를 프로세싱 소프트웨어(예: Imaris)로 가져옵니다.</li>
	<li>인접한 핵을 배제하기 위해 수동 세분화에 의해 핵을 정의한다. Coloc 소프트웨어를 사용합니다. 2형형형량 사이의 전체 부피 내에서 Pearson의 상관계수(PcC)를 수집합니다.</li>
</ol>

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저자는 공개 할 것이 없습니다.

이 프로토콜은 전체 마운트 성인 Drosophila 고환의 성공적인 면역 라벨링을위한 방법을 제공합니다. 그밖 절차에서 보고된 바와 같이 젊은 남성은 정자를 방해하는 리스크가 있는 발달 정액의 존재를 극소화하기 위하여 (우리는 심지어 백과기 후에 20 시간 미만으로 나이를 단축했습니다) 이용되어야 합니다. 해부는 신속해야 하며 항체 희석은 신호 대 잡음비(21,<sup class="xr...

드로소필라 고환은 핵 건축 연구를 위한 귀중한 모델입니다. 미토틱 정자 세포에서 핵 부피는 주로 크로마틴에 의해 점유됩니다. 이 세포는 16개의 1차 정자세포의 낭종을 생성하는 분열의 4라운드를 겪습니다. 다음 며칠 동안, 정자 세포는 6 개의 발달 단계 (S1-S6)를 통과하며, 부피가 크게 증가하여 20 배1,2. 그들은 또한 그들의 핵 형태를 변경. 라빈 Dm0에 의해 드러난 핵의 윤곽은 불규칙해지고 깊은 질1,3을나타낸다. 이는 크로마틴 조직1,4,5,6,7에서광범위한 유전자 발현 변화 및수정을 수반한다. 단계 간 염색체는 단계 S4-S5에서 잘 정의되어 있으며, 2개의 주요 이중성 자가솜과 뉴클레오투스로 도킹된 X-Y 쌍에 대응하는 3개의 뚜렷한 질량을 형성한다.
Mad1은 원래 미토틱 체크포인트의 핵심 성분으로격리되었습니다(8에서검토됨). 상간에서, 핵봉투에 주로 위치하는 것으로 설명되고 핵9,10,11에있다. 드로소필라 고환에서, 우리는 Mad1이 핵에 두드러진 것을 보고했습니다, 밀접하게 두 개의 주요 염색체 염색체질량과 연결되고 XY 크로마틴과 더 적은 정도에. 우리는 이 크로마틴 관련 구조를 민트(Mad1 함유 핵 영토)12로정의하였다. 네 가지 다른 단백질은 정자 세포에서 MINTs와 연관되었다: 뉴클레오포린 Mtor/Tpr, SUMO 펩티다아제 Ulp1, 미토틱 체크포인트 단백질 Mad2 및 폴리콤 과 같은 복합 Raf212의서브유닛.
MINTs의 설명을 완료하기 위하여는, 우리는 항체를 이용해야 하고 일반적으로 고환에 있는 면역 염색과 가진 기술적인 문제에 직면했습니다: 고환의 깊이에 얼룩의 중요한 비 균일성의 결과로, 특히 큰 정자 세포에. Squashed 준비는 항체 접근을 증가시켰지만 압축 된 이미지로 이어졌고, 3D 분석을 제한하고, 공동화를 포함한정량형 형광의 측정을 방지했다. 항체 침투의 문제는 또한 0.3% Na deoxycholate13과같은 permeabilizing 에이전트에 의해 해결될 수 있었습니다, 그러나 이 절차는 우리의 손에 재현가능한 결과를 산출하지 않았습니다. 우리는 많은 공동 라벨 링 실험을 수행했기 때문에, 우리는 permeabilization 프로토콜을 개선하기 위해 보았다. 1% NP40 플러스 헵탄의 조합은 특히 큰 정자 세포를 연구하는 데 더 많은 재현성을 초래했습니다.
이 프로토콜은 서스펜션에서 고환의 고정 및 면역 스테인링을 수행하여 샘플 품질을 개선하고 재현성을 향상시키고 공초점 현미경 검사에 적합합니다. 우리는 큰 정자 세포의 핵 폐색 구조와 특정 핵 봉투 단백질의 공간 관계를 더 잘 정의하기 위해 프로토콜을 사용했습니다. 이 방법은 예를 들어 크로마틴, 핵염 및 핵 모공을 포함한 핵의 동적 구조적 변화와 정자 세포 발달을 수반하는 변화에 대한 더 나은 조사를 허용할 것이다.

<table><tbody><tr><td>&lt;em&gt;&lt;strong&gt;Drosophila srains&lt;/strong&gt;&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>ketelGFP y; ketelGFP/y+CyO</td><td></td><td></td><td>Gift from A. Debec (Institut Jacques Monod, UMR 7592, Paris, France)</td></tr><tr><td>Mad1-GFP</td><td></td><td></td><td>J Cell Sci. 2011 May 15;124(Pt 10):1664-71.</td></tr><tr><td>Mtor RNAi&amp;nbsp;</td><td>VDRC</td><td>ID 110218</td><td>Vienna Drosophila RNAi Center</td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DAPI&amp;nbsp;</td><td>Thermo</td><td>D1306</td><td>Stock solution must be prepared in H2O</td></tr><tr><td>Dumont # 5 Fine Forseps&amp;nbsp;</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>MBP Wide Bore Pipette Tips</td><td>Fisherbrand</td><td>11917744</td><td>Wide aperture tip</td></tr><tr><td>Thermo Scientific&amp;nbsp;0.2 mL Individual Tube</td><td>Thermo</td><td>AB-0620</td><td></td></tr><tr><td>2 ml micro centrifuge tubes&amp;nbsp;</td><td>Sigma</td><td>T3531-200EA&amp;nbsp;</td><td>Siliconized 2ml tube</td></tr><tr><td>Citifluor AF1</td><td>Citifluor</td><td>AF1</td><td></td></tr><tr><td>Dakopen&amp;nbsp;</td><td>Sigma</td><td>Z377821-1EA&amp;nbsp;</td><td></td></tr><tr><td>Normal Goat Serum (NGS)</td><td>Sigma</td><td>NS02L-1ML</td><td></td></tr><tr><td>Paraformaldehyde 4%</td><td>Sigma</td><td>P6148-500G&amp;nbsp;</td><td>Paraformaldehyde (4%) dissolved in PBS 1X&amp;nbsp;; aliquot by 600 ml&amp;nbsp;</td></tr><tr><td>PBS 1X</td><td>Thermo</td><td>14190094</td><td>DPBS, no calcium, no magnesium</td></tr><tr><td>0.1% (0.2 or 0.3%) PBS-T&amp;nbsp;</td><td></td><td></td><td>PBS 1X&amp;nbsp; containing 0.1% (0.2 % or 0.3%) Triton X-100</td></tr><tr><td>Triton X-100, for molecular biology,&amp;nbsp;</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GFP booster</td><td>Chromotek</td><td>gba488</td><td>1/200</td></tr><tr><td>Mouse anti-Mtor</td><td></td><td></td><td>1/40 gift from J. Johansen, Iowa State University</td></tr><tr><td>Rat anti-Nup62</td><td></td><td></td><td>1/200 gift from H. Ohkura (University of Edinburgh, UK</td></tr><tr><td>Guinea-pig anti-Ulp1</td><td></td><td></td><td>1/200 gift from C. P. Verrijzer, Erasmus University, Rotterdam</td></tr><tr><td>Rabbit anti-Raf2</td><td></td><td></td><td>1/200 gift from C. P. Verrijzer, Erasmus University, Rotterdam</td></tr><tr><td>DyLight conjugated series&amp;nbsp;</td><td>Thermo</td><td></td><td>1/500 Donkey anti-(guinea-pig, mouse,&amp;nbsp; rabbit or rat) as second antibodies</td></tr></tbody></table>

immunostaining of whole-mount drosophila testes for 3d confocal analysis of large spermatocytes

Drosophila 고환은 줄기 세포 생물학을 포함하여 생물학 프로세스를 공부하기 위한 강력한 모형 시스템, 핵 건축, meiosis 및 정액 발달. 그러나, 전체 Drosophila 고환의 면역 라벨링은 항체 침투로 인한 염색의 중요한 비 균일성과 수시로 연관된다. 분석의 3D 품질을 감소하기 때문에 분쇄 된 준비는 부분적으로 문제를 부분적으로 극복합니다. 본 명세서에서는 액체 매체의 면역 라벨링과 함께 고정 시 NP40 및 heptane을 사용하여 전체 마운트 프로토콜을 설명합니다. 그것은 재현 가능하고 신뢰할 수있는 라벨과 함께 공초점 현미경 검사법에 적합한 볼륨을 보존합니다. 우리는 공초점 섹션에서 정자 세포 핵의 3D 재구성의 다른 예를 보여줍니다. 장 내 및 고환 간 재현성은 상이한 유전자형으로부터 단일 세포 간의 형광의 3D 정량화 및 비교를 허용합니다. 우리는 핵 내 MINT 구조 (Mad1 함유 인트라 핵 영토)의 다른 구성 요소뿐만 아니라 핵 모공 복합체와 관련된 두 가지 구성 요소를 사용하여 이 프로토콜과 시험의 가장 큰 세포인 S4-S5 정자 세포에 대한 응용 프로그램을 설명했습니다.

Drosophila 고환의 적당한 얼룩을 얻기에 있는 주요 장애물은 항체의 제한된 침투성입니다, 이는 부분적으로 분쇄된 준비를 사용하여 서 그러나 3D 분석의 제한을 유도하는 대가로 (예를 들면 3D 표현 또는 공동화의 측정)를 유도해서 부분적으로 해결되었습니다. 절차는 균일 한 라벨을 허용하고 고환의 모든 세포의 볼륨을 보존. 여기에서 우리는 S4-S5 정자 세포의 3D 표현에 방법의 그림을 집중했습니?...

Watch this Scientific Journal Video about Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes at JoVE.com

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

우리는 여기에 공초점 현미경 검사법에 적합하고 재현 가능하고 신뢰할 수있는 라벨을 허용 Drosophila 고환의 전체 마운트 준비 및 면역 스테인링을위한 향상된 프로토콜을 제시합니다. 우리는 대형 S4-S5 정자 세포에 대한 공동 지역화 실험의 3D 표현 및 정량화에 의해이 프로토콜을 설명합니다.

대형 정자 세포의 3D 공초점 분석을 위한 전산 Drosophila 고환의 면역 염색

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm ...

immunostaining-whole-mount-drosophila-testes-for-3d-confocal-analysis

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JoVE Journal

Biology

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

4.1K 조회수.  Université de Paris, F 75006, Paris, France. 우리는 여기에 공초점 현미경 검사법에 적합하고 재현 가능하고 신뢰할 수있는 라벨을 허용 Drosophila 고환의 전체 마운트 준비 및 면역 스테인링을위한 향상된 프로토콜을 제시합니다. 우리는 대형 S4-S5 정자 세포에 대한 공동 지역화 실험의 3D 표현 및 정량화에 의해이 프로토콜을 설명합니다.

비디오: Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

Upright Imaging of Drosophila Egg Chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

의 수직 이미지<em&gt; 초파리</em&gt; 계란 챔버

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

연구

발생학

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

살아있는, 그대로 조직에서 세포 분열의 분석을 위한 초파리 애벌레와 Pupal 고환의 준비

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...

Whole Ovary Immunofluorescence, Clearing, and Multiphoton Microscopy for Quantitative 3D Analysis of the Developing Ovarian Reserve in Mouse

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells entering meiotic prophase I are eliminated during Fetal Oocyte Attrition (FOA) and the first week of postnatal life. Three major mechanisms regulate the number of oocytes that survive during development and establish the ovarian reserve in females entering puberty. In the first wave of oocyte loss, 30-50% of the oocytes are eliminated during early FOA, a phenomenon that is attributed to high Long interspersed nuclear element-1 (LINE-1) expression. The second wave of oocyte loss is the elimination of oocytes with meiotic defects by a meiotic quality checkpoint. The third wave of oocyte loss occurs perinatally during primordial follicle formation when some oocytes fail to form follicles. It remains unclear what regulates each of these three waves of oocyte loss and how they shape the ovarian reserve in either mice or humans.
Immunofluorescence and 3D visualization have opened a new avenue to image and analyze oocyte development in the context of the whole ovary rather than in less informative 2D sections. This article provides a comprehensive protocol for whole ovary immunostaining and optical clearing, yielding preparations for imaging using multiphoton microscopy and 3D modeling using commercially available software. It shows how this method can be used to show the dynamics of oocyte attrition during ovarian development in C57BL/6J mice and quantify oocyte loss during the three waves of oocyte elimination. This protocol can be applied to prenatal and early postnatal ovaries for oocyte visualization and quantification, as well as other quantitative approaches. Importantly, the protocol was strategically developed to accommodate high-throughput, reliable, and repeatable processing that can meet the needs in toxicology, clinical diagnostics, and genomic assays of ovarian function.

Here, we present an optimized protocol for imaging entire ovaries for quantitative and qualitative analyses using whole-mount immunostaining, multiphoton microscopy, and 3D visualization and analysis. This protocol accommodates high-throughput, reliable, and repeatable processing that is applicable for toxicology, clinical diagnostics, and genomic assays of ovarian function.

Female fertility and reproductive lifespan depend on the quality and quantity of the ovarian oocyte reserve. An estimated 80% of female germ cells ...

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 ...

생물학

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 Centrosomes in Fly Testes

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell differentiation. Centrosomes are essential to determine the cell division axis during mitosis and to nucleate cilia during interphase. The identity of the proteins that mediate these dynamic changes remains only partially known, and the function of many of the proteins that have been implicated in these processes is still rudimentary. Recent work has shown that Drosophila spermatogenesis provides a powerful system to identify new proteins critical for centrosome function and formation as well as to gain insight into the particular function of known players in centrosome-related processes. Drosophila is an established genetic model organism where mutants in centrosomal genes can be readily obtained and easily analyzed. Furthermore, recent advances in the sensitivity and resolution of light microscopy and the development of robust genetically tagged centrosomal markers have transformed the ability to use Drosophila testes as a simple and accessible model system to study centrosomes. This paper describes the use of genetically-tagged centrosomal markers to perform genetic screens for new centrosomal mutants and to gain insight into the specific function of newly identified genes.

Imaging of centrosomal proteins during Drosophila spermatogenesis is a powerful method to identify new proteins critical for centrosome biology as well as to elucidate the particular function of known players in this process.

비행 고환에있는 영상 중심체

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

정자의 세포 학적 분석 : 라이브와 고정 준비<em&gt; 초파리</em&gt; 고환

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and organogenesis. Immunostaining allows for the visualization of developing tissues and organs. However, a protective cuticle that forms at the end of embryogenesis prevents permeation of antibodies into late-stage embryos and larvae. While dissection prior to immunostaining is regularly used to analyze Drosophila larval tissues, it proves inefficient for some analyses because small tissues may be difficult to locate and isolate. Sonication provides an alternative to dissection in larval Drosophila immunostaining protocols. It allows for quick, simultaneous processing of large numbers of late-stage embryos and larvae and maintains in situ morphology. After fixation in formaldehyde, a sample is sonicated. Sample is then subjected to immunostaining with antigen-specific primary antibodies and fluorescently labeled secondary antibodies to visualize target cell types and specific proteins via fluorescence microscopy. During the process of sonication, proper placement of a sonicating probe above the sample, as well as the duration and intensity of sonication, is critical. Additonal minor modifications to standard immunostaining protocols may be required for high quality stains. For antibodies with low signal to noise ratio, longer incubation times are typically necessary. As a proof of concept for this sonication-facilitated protocol, we show immunostains of three tissue types (testes, ovaries, and neural tissues) at a range of developmental stages.

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.

늦게 단계 배아와 애벌레의 초음파 - 촉진 면역 형광 염색<em&gt; 초파리</em&gt; 조직<em&gt; 제자리에서</em

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and ...

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 ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

세포 전 형 구조의 슈퍼 해상도 라이브 세포 이미징

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

대형 정자 세포의 3D 공초점 분석을 위한 전산 Drosophila 고환의 면역 염색

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