Name: dissection and staining of drosophila pupal ovaries
Uploaded: 2018-03-02T14:30:00
Description: Watch this Scientific Journal Video about Dissection and Staining of Drosophila Pupal Ovaries at JoVE.com

この研究は、国立衛生研究所によって支えられた (d. k. に GM079351 RO1)。ドロテア Godt に彼女の元のプロトコルに基づいて蛹卵巣の解剖に彼女の有用なアドバイスありがちましょう。我々 はまた、彼女の援助と原稿のコメントをエイミー Reilein を感謝します。

1. 仔
<ol><li>10 の男性と 15 女性ショウジョウバエ成虫酵母を添加した通常豊かなフライ食品の瓶で目的遺伝子の約を組み合わせます。バイアルを過密を避けるため、もはや 2-4 h14以上の卵を産む雌成虫の交尾を許可します。</li>
	<li>はえの食糧と異なるバイアルに対して開くバイアルをタップして、大人をバイアルから新しい瓶に転送します。3-4 日間室温で幼虫に成?...

<ol>
	<li>Losick, V., Morris, L., Fox, D., Spradling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Stem+Cell+Niches:+A+Decade+of+Discovery+Suggests+a+United+View+of+Stem+Cell+Regulation.">Drosophila Stem Cell Niches: A Decade of Discovery Suggests a United View of Stem Cell Regulation.</a> Dev Cell. 21 (1), 159-171 (2011).</li><li>Kirilly, D., Xie, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+ovary:+an+active+stem+cell+community.">The Drosophila ovary: an active stem cell community.</a> Cell Res. 17 (1), 15-25 (2007).</li><li>Dansereau, D. A., Lasko, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Development+of+Germline+Stem+Cells+in+Drosophila.">The Development of Germline Stem Cells in Drosophila.</a> Method Mol Biol. 450, 3-26 (2008).</li><li>Pearson, J., L&#243;pez-Onieva, L., Rojas-R&#237;os, P., Gonz&#225;les-Reyes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+Drosophila+stem+cell+biology.">Recent advances in Drosophila stem cell biology.</a> Int J Dev Biol. 53, 1329-1339 (2009).</li><li>Arwert, E. N., Hoste, E., Watt, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+stem+cells,+wound+healing+and+cancer.">Epithelial stem cells, wound healing and cancer.</a> Nat Rev Cancer. 12 (3), 170-180 (2012).</li><li>Barker, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crypt+stem+cells+as+the+cells-of-origin+of+intestinal+cancer.">Crypt stem cells as the cells-of-origin of intestinal cancer.</a> Nature. 457 (7229), 608-611 (2009).</li><li>Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+myeloid+leukemia:+proving+ground+for+cancer+stem+cells.">Chronic myeloid leukemia: proving ground for cancer stem cells.</a> Cell. 119 (3), 314-316 (2004).</li><li>Reilein, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+direct+stem+cell+derivatives+defined+by+stem+cell+location+and+graded+Wnt+signaling.">Alternative direct stem cell derivatives defined by stem cell location and graded Wnt signaling.</a> Nat Cell Biol. 19 (5), 433-444 (2017).</li><li>Eliazer, S., Buszczack, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finding+a+niche:+studies+from+the+Drosophila+ovary.">Finding a niche: studies from the Drosophila ovary.</a> Stem Cell Res Ther. 2 (6), 45 (2011).</li><li>Godt, D., Laski, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+cell+rearrangement+and+cell+recruitment+in+Drosophila+ovary+morphogenesis+and+the+requirement+of+bric+&#224;+brac.">Mechanisms of cell rearrangement and cell recruitment in Drosophila ovary morphogenesis and the requirement of bric &#224; brac.</a> Development. 121 (1), 173-187 (1995).</li><li>King, R. C., Aggarwal, S. K., Aggarwal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+the+female+Drosophila+reproductive+system.">The development of the female Drosophila reproductive system.</a> J Morphol. 124 (2), 143-166 (1968).</li><li>Vlachos, S., Jangam, S., Conder, R., Chou, M., Nystul, T., Harden, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Pak-regulated+cell+intercalation+event+leading+to+a+novel+radial+cell+polarity+is+involved+in+positioning+of+the+follicle+stem+cell+niche+in+the+Drosophila+ovary.">A Pak-regulated cell intercalation event leading to a novel radial cell polarity is involved in positioning of the follicle stem cell niche in the Drosophila ovary.</a> Development. 142 (1), 82-91 (2015).</li><li>Irizarry, J., Stathopoulos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF+signaling+supports+Drosophila+fertility+by+regulating+development+of+ovarian+muscle+tissues.">FGF signaling supports Drosophila fertility by regulating development of ovarian muscle tissues.</a> Dev Biol. 404 (1), 1-13 (2015).</li><li>Maimon, I., Gilboa, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+and+Staining+of+Drosophila+Larval+Ovaries.">Dissection and Staining of Drosophila Larval Ovaries.</a> J Vis Exp. (51), e2537 (2011).</li><li>Kerkis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Growth+of+the+Gonads+in+DROSOPHILA+MELANOGASTER.">The Growth of the Gonads in DROSOPHILA MELANOGASTER.</a> Genetics. 16 (3), 212-224 (1931).</li><li>Yamanaka, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroendocrine+Control+of+Drosophila+Larval+Light+Preference.">Neuroendocrine Control of Drosophila Larval Light Preference.</a> Science. 341 (6150), 1113-1116 (2013).</li><li>Bainbridge, S., Bownes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staging+the+metamorphosis+of+Drosophila+melanogaster.">Staging the metamorphosis of Drosophila melanogaster.</a> Development. 66, 57-80 (1981).</li><li>Courdec, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bric+&#224;+brac+locus+consists+of+two+paralagous+genes+encoding+BTB/POZ+domain+proteins+and+acts+as+a+homeotic+and+morphogenetic+regulator+of+imaginal+development+in+Drosophila.">The bric &#224; brac locus consists of two paralagous genes encoding BTB/POZ domain proteins and acts as a homeotic and morphogenetic regulator of imaginal development in Drosophila.</a> Development. 129, 2419-2433 (2002).</li><li>Arrese, E. L., Soulages, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=INSECT+FAT+BODY:+ENERGY,+METABOLISM,+AND+REGULATION.">INSECT FAT BODY: ENERGY, METABOLISM, AND REGULATION.</a> Annu Rev Entomol. 55, 207-225 (2011).</li><li>Zhang, Y., Xi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fat+Body+Development+and+its+Function+in+Energy+Storage+and+Nutrient+Sensing+in+Drosophila+melanogaster.">Fat Body Development and its Function in Energy Storage and Nutrient Sensing in Drosophila melanogaster.</a> J Tissue Sci Eng. 6 (1), (2014).</li><li>Wong, L. C., Schedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+Drosophila+Ovaries.">Dissection of Drosophila Ovaries.</a> J Vis Exp. (1), e52 (2006).</li></ol>

このプロトコルの最も重要かつ困難なステップには、固定する前に蛹卵巣の準備が含まれます。抗体と、卵巣、小さくて蛹腹部内脂肪体細胞によって埋められること十分に汚れることを確認、鉗子、腹部の袋に大きな開口部を引き裂くだけでなくが、また妨げる脂肪体細胞を抽出することが重要です、抗体からの卵巣。この手順が正常に実行が必要です腹部の袋から脂肪体細胞を洗いながら?...

ショウジョウバエの卵巣を用いた幹細胞研究は、幹細胞ニッチ1,2,3,4の最初のドキュメンテーションから広く展開しています。リネージュ トレース遺伝的ツール、解剖がよく幹細胞を研究に使用されているショウジョウバエ卵巣の開発と幹細胞維持、増殖、および幹細胞ニッチの運命を制御するシグナルします。これらのシグナル伝達経路の知識は、異常な幹細胞活動5,6,7から由来する癌の潜在的な原因への洞察力をもたらす可能性があります。また、最近、卵胞幹細胞 (Fsc)、として知られているショウジョウバエ卵巣の体性幹細胞、彼らの組織の8の多くの側面で哺乳類の腸管幹細胞のように強く示されています。このため、ショウジョウバエ卵巣が幹細胞の挙動を研究するための非常に有用なモデル システムです。
幼虫及び成虫の卵巣はそれぞれ初期の幹細胞の開発とニッチで最終的な幹細胞組織への手がかりを提供、蛹卵巣は生殖細胞と体細胞で再編成、彼らのアイデンティティを確立中間構造9,10. 分化と空間構成に関する質問のままいくつかの研究は、蛹卵巣10、11,12,13組織開発の側面を検討しているが蛹の発育中の卵巣細胞型。特に、Fsc の仕様は、この期間中に発生します。このプロトコルの概要を解剖の目的の時点で蛹卵巣染色法-蛹卵巣における大人の段階に幼虫から詳細を分析時間コース実験で使用できるテクニック。
小型、半透明、蛹卵巣蛹腹部内の到達不能にする、このプロトコルは抗体アクセスを妨げる腹部脂肪体組織を除去するカスタムメイドの薄い先端パスツール ピペットなどのツールを利用して、卵巣。蛹の"Nutator"の卵巣を揺動の穏やかなプラットフォームを提供しています高い視認性を汚す抗体中に使用される明確なチェンバード coverglassMaimon、ギルボア14による幼虫卵巣の解剖のプロトコルに基づいて、トリトン X-100 の比較的高い濃度で採用されている染色の手順の最初の手順に細胞膜透過と抗体アクセスを最大化する、卵巣細胞。

<table>
	<tbody>
		<tr>
			<td>Dumont #5 Forceps, biology</td>
			<td>Fine Scientific Tools</td>
			<td>11252-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc Lab-Tek Chambered Coverglass</td>
			<td>Thermo Fisher Scientific</td>
			<td>155383</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Nikon</td>
			<td>SMZ-10A</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM 700</td>
			<td></td>
		</tr>
		<tr>
			<td>Analysis software</td>
			<td>Carl Zeiss</td>
			<td>Zen</td>
			<td></td>
		</tr>
		<tr>
			<td>9 Depression Glass Spot Plates</td>
			<td>Pyrex</td>
			<td>7220-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipet</td>
			<td>Fisher Scientific</td>
			<td>13-678-6B</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipet bulb</td>
			<td>Various vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td>Various vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherfinest Premium Frosted Microscope Slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>12-544-2</td>
			<td></td>
		</tr>
		<tr>
			<td>22 x 22 mm glass coverslips No 1</td>
			<td>VWR</td>
			<td>48366-067</td>
			<td></td>
		</tr>
		<tr>
			<td>Dapi Fluoromount-G</td>
			<td>SouthernBiotech</td>
			<td>0100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Double-sided tape</td>
			<td>Scotch</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nutator</td>
			<td>Clay Adams</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine brush #0, #3-#5</td>
			<td>Various vendors</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gilson Pipetman Starter Kit</td>
			<td>Thomas Scientific</td>
			<td>F167300</td>
			<td>Contains p20, p200, p1000 pipettors</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Dilute to 4% paraformaldehyde in 1x PBS</td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>Sigma-Aldrich</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Ambion</td>
			<td>AM9624</td>
			<td>Dilute to 1x PBS</td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Jackson ImmunoResearch</td>
			<td>5000121</td>
			<td>Dilute to 10% normal goat serum in PBST with 0.5% Triton concentration</td>
		</tr>
		<tr>
			<td>Primary antibodies (in protocol: 7G10 anti-Fasciclin III diluted 1:250, rabbit anti-phosphohistone H3 diluted 1:1000)</td>
			<td>Various vendors (in protocol: Developmental Studies Hybridoma Bank, Millipore)</td>
			<td></td>
			<td>Dilute in PBST with 0.5% Triton concentration</td>
		</tr>
		<tr>
			<td>Secondary antibodies (in protocol: Alexa-546, FITC-conjugated anti-rabbit serum)</td>
			<td>Various vendors (in protocol: Molecular Probes, Jackson ImmunoResearch Laboratories, Inc.)</td>
			<td></td>
			<td>Dilute in PBST with 0.5% Triton concentration</td>
		</tr>
		<tr>
			<td>Fly vials</td>
			<td>Denville Scientific</td>
			<td>V9406</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Balls, For Wide Vials</td>
			<td>Genesee Scientific</td>
			<td>51-102W</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast, Bakers Dried Active</td>
			<td>MP Biomedicals</td>
			<td>101400</td>
			<td></td>
		</tr>
		<tr>
			<td>Fly food</td>
			<td>Produced in laboratory</td>
			<td></td>
			<td>Mixture of water, brewer&#39;s yeast, cornmeal, molasses, agar, EtOH, penicillin, methyl 4-hydrobenzoate, and propionic acid</td>
		</tr>
		<tr>
			<td>Male and female Drosophila flies (genotype used in protocol: yw; P[Fz3-RFP, w+]/TM2)</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection and staining of drosophila pupal ovaries

大人のショウジョウバエ卵巣とは異なり蛹卵巣は比較的アクセスし、彼らの小型のため確認することは困難、サイズ、半透明、蛹のケース内で被覆。蛹卵巣の解剖の課題はまた蛹内の物理的な場所にある: 卵巣蛹腹部内脂肪体細胞に囲まれているし、これらの脂肪細胞は適切な抗体の汚損のため許可を削除必要があります。これらの課題を克服するためには、このプロトコルは、蛹の腹部からの脂肪体細胞を抽出するためのカスタマイズされたパスツール pipets を利用しています。また、蛹の可視性を改善するために染色のプロセス中に、カバーグラスチェンバーを遠心管の代わりに使用します。ただし、これらとこのプロトコルで使用するツールの他の利点にもかかわらず、これらの技術の実行が成功は、まだ蛹卵巣のサイズが小さいため練習のいくつかの日を伴うことがあります。このプロトコルに記載されているテクニックは、蛹の開発のさまざまな段階で卵巣を解析時間コース実験に適用でした。

この手順が正常に実行明確な抗体の汚損構造とショウジョウバエ蛹卵巣の細胞組織を明らかにする必要があります。このプロトコルで説明されている免疫組織化学は、幼生及び成体卵巣における一般染色の種類の細胞を識別するために使用できます。派生した蛹の茎の細胞群細胞18 (白ファシクリン III で説明します)、図 3</stron...

Watch this Scientific Journal Video about Dissection and Staining of Drosophila Pupal Ovaries at JoVE.com

Dissection and Staining of Drosophila Pupal Ovaries

ショウジョウバエ卵巣は、幹細胞ニッチの開発を研究するためのエクセレント モデル システムです。幼虫及び成虫の卵巣を解剖するための方法を公開されている、蛹卵巣解剖詳しく公開されていない別のテクニックが必要です。ここでは、解離性、染色、および蛹卵巣をマウントするためのプロトコルを概説します。

郭清およびショウジョウバエ蛹卵巣の汚損

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Research

JoVE Journal

Developmental Biology

Dissection and Staining of Drosophila Pupal Ovaries

Live-imaging of the Drosophila Pupal Eye

Live-imaging of the Drosophila Pupal Eye

のライブイメージング<em&gt;ショウジョウバエ</em&gt;蛹アイ

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

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

文化と共培養マウスの卵巣と卵胞

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Dissection and Culture of Mouse Embryonic Kidney

マウス胎児腎臓の解剖および培養

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

Immunodetections または PCR の試金に適したショウジョウバエの蛹の翼を解剖する迅速かつ効率的な方法

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

郭清ときのこ体の免疫蛍光染色と大人のショウジョウバエ脳光受容器ニューロン

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

全体のマウントの蛍光抗体法と培養マウス卵巣の卵胞の定量化

Research in the field of mammalian reproductive biology often involves evaluating the overall health of ovaries and testes. Specifically, in females, ...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

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

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

解凍、養殖、ショウジョウバエ細胞をトウキョウ

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

プパル期のショウジョウバエ上皮の組織オリエンテーションと成長ダイナミクスのイメージングと解析

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

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

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

生きた無傷組織における細胞分裂解析のためのショウジョウバエ・ラーバルとプパル精巣の調製

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