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

这项研究得到了国家卫生研究院 (RO1 GM079351 到交流) 的支持。我们感谢 Godt 为她提供了有益的建议, 蛹卵巢解剖根据她的原始协议。我们还感谢艾米 Reilein 对手稿的帮助和评论。

1. EggLaying
<ol><li>结合大约十个男性和十五个女性成年果蝇的期望基因型的苍蝇在一瓶正常丰富的苍蝇食品补充酵母。为了避免瓶子过度拥挤, 允许交配雌性产卵不长于 2–4 h14。</li>
	<li>把这些成年人从瓶子里移到一个新瓶子里, 然后用小瓶子打开, 用苍蝇食物来对付不同的瓶子。允许卵在室温下发育成幼虫, 3–4天。</li>
</ol>2. 选择雌幼虫
<ol><li>使用柔软...

<ol>
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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. 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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>

本议定书最关键和最困难的步骤是在固定前准备蛹卵巢。为了确保卵巢, 小和埋在蛹腹部的脂肪体细胞, 有足够的抗体, 这是重要的, 不仅撕裂一个大的开放, 在腹腔麻袋与钳, 但也提取脂肪体细胞, 阻碍卵巢的抗体。成功执行此步骤需要在巴斯德吸管灯泡上应用微妙的压力, 同时将脂肪体细胞从腹袋中清洗出来 (例如, 在这一步中, 在每秒的时间内都应留出腹部)。如果不使用温和的力量, 可能会...

干细胞研究使用果蝇卵巢已广泛扩展自第一个文件的干细胞利基1,2,3,4。随着沿袭追踪遗传工具的发展,果蝇卵巢解剖通常被用来研究干细胞的谱系和信号通路, 调节干细胞的维持、增殖和命运。有关这些信号通路的知识可能会对源于异常干细胞活动的癌症的潜在病因产生洞察力5,6,7。最近还显示, 在果蝇卵巢中, 称为卵泡干细胞 (开办) 的体细胞干细胞在其组织的许多方面都与哺乳动物的肠道干细胞有很大的相似性, 这些细胞有8。因此,果蝇卵巢是研究干细胞行为的一个非常有用的模型系统。
幼虫和成人卵巢为早期干细胞的发育和生态位的最终干细胞组织提供了线索, 蛹卵巢是生殖和体细胞重组和建立其身份的中间结构。9,10. 虽然有几项研究已经检查了蛹子房10、11、12、13的组织发展方面, 但关于分化和空间组织的问题依然存在。蛹发育期间的卵巢细胞类型。特别是, 开办的规格在这个期间发生。本协议概述了在所需的时间点解剖和染色蛹卵巢的方法, 这一技术可用于从幼虫到成年期详细分析蛹卵巢发育的时间过程实验。
为了解释蛹腹中蛹卵巢的体积小、半透明和难以接近的问题, 本协议利用诸如自定义的薄尖巴斯德吸管等工具去除腹部脂肪体组织阻碍抗体进入卵巢.在抗体染色过程中使用的一个清晰的腔内 coverglass, 可以使蛹的可见度更高, 并且可以在 "Nutator" 上摇动卵巢的温和平台。基于 Maimon 和基利波山14的幼虫卵巢解剖协议, 在染色过程的最初步骤中采用了相对较高浓度的海卫 X-100, 以最大限度地提高细胞膜通透和抗体进入卵巢细胞。

<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

与成年的果蝇卵巢不同, 蛹卵巢由于其体积小、半透明和在蛹的情况下被包裹而难以进入和检查。解剖蛹卵巢的挑战还在于它们在蛹中的物理位置: 卵巢被蛹腹部内的脂肪体细胞所包围, 必须除去这些脂肪细胞, 以允许适当的抗体染色。为了克服这些挑战, 本协议利用自定义的巴斯德滴管从蛹腹部提取脂肪体细胞。此外, 在染色过程中使用腔内 coverglass 代替离心管, 以提高蛹的可见度。然而, 尽管本议定书使用的工具有这些和其他优点, 但由于蛹卵巢体积小, 成功地执行这些技术可能还需要几天的实践。本议定书所概述的技术可应用于蛹发育不同阶段的卵巢分析的时间过程实验。

成功执行此过程应导致清晰的抗体染色, 揭示了一个果蝇蛹卵巢的结构和细胞组织。免疫组织化学在本议定书中概述可用于识别常见的细胞类型在幼虫和成人卵巢染色。从群单元派生的蛹茎细胞18 (由白色 Fasciclin III 所概述) 显示在图 3中。除了突出蛹卵巢的细胞组织外, 特定于细胞增殖的抗体染色 (如以绿色显示的<strong class="xf...

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

Dissection and Staining of Drosophila Pupal Ovaries

果蝇卵巢是研究干细胞小生境发育的优良模型系统。虽然已经公布了解剖幼虫和成人卵巢的方法, 蛹卵巢解剖需要不同的技术, 没有详细发表。在这里, 我们概述了解剖, 染色和安装蛹卵巢的协议。

果蝇蛹卵巢的解剖与染色

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

实时成像<em&gt;果蝇</em&gt;蛹眼

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

科研

发育生物学

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

文化和共同培养小鼠的卵巢和卵巢卵泡

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

小鼠胚胎肾的解剖与培养

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

一种快速高效的解剖蛹翅的方法, 适用于 Immunodetections 或 PCR 检测

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

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

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

培养小鼠卵巢的全贴装免疫荧光和卵泡定量研究

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

嗜酸性粒植物视网膜免疫组织化学、西方分析和 Rna 分离的解剖

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

解冻、培养和冷冻保存的嗜酸性粒细胞系

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

普帕尔阶段发育性皮瘤中组织定向和生长动力学的成像与分析

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

用于活、完整组织中细胞分部分析的果蝇拉瓦尔和普帕尔睾试验的制备

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