Name: a rapid and efficient method to dissect pupal wings of drosophila suitable for immunodetections or pcr assays
Uploaded: 2017-12-30T14:00:00
Description: Watch this Scientific Journal Video about A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays at JoVE.com

我们要感谢工对这个项目的财政支持, 对玛丽安娜 Di Doménico 为她的技术援助与共焦显微镜;Báez 为他的手稿修正;为他献身于创造视频的科雷亚;Rosano 为视频和布卢明顿果蝇股票中心提供了本研究中使用的股票的声音。Apa1 单克隆抗 Ptc 开发的伊莎贝尔格雷罗是从发展研究杂交瘤银行 (DSHB) 的主持下, 研究院和维护由爱荷华大学, 生物科学系, 爱荷华市 IA 52242。

1天
1. 收集和培养蛹。
<ol>
	<li>在蛹形成 (APF) 或蛹后, 使用湿的画笔从培养的小瓶中取出0小时, 并将它们放入新的瓶子中, 以几乎完成开发期 (见2.1 和 2.2)。 
	注意:应该使用标准的培养规程来维护健康的苍蝇种群。在产卵三或四天后, 成人可以从小瓶中取出, 以使个体得到最佳的发育。他们保持在恒定的温度, 直到第三个幼虫开始蛹。幼虫/蛹的转变是以蛹的形成为...

<ol>
	<li>Serrano, N., O'Farrell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limb+morphogenesis:+connections+between+patterning+and+growth.">Limb morphogenesis: connections between patterning and growth.</a> Curr Biol. 7 (3), R186-R195 (1997).</li><li>Blair, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartments+and+appendage+development+in+Drosophila.">Compartments and appendage development in Drosophila.</a> Bioessays. 17 (4), 299-309 (1995).</li><li>Klein, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wing+disc+development+in+the+fly:+the+early+stages.">Wing disc development in the fly: the early stages.</a> Curr Opin Genet Dev. 11 (4), 470-475 (2001).</li><li>Beira, J. V., Paro, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+legacy+of+Drosophila+imaginal+discs.">The legacy of Drosophila imaginal discs.</a> Chromosoma. 125 (4), 573-592 (2016).</li><li>De Celis, J. F., Diaz-Benjumea, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+basis+for+vein+pattern+variations+in+insect+wings.">Developmental basis for vein pattern variations in insect wings.</a> Int J Dev Biol. 47 (7-8), 653-663 (2003).</li><li>Blair, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wing+vein+patterning+in+Drosophila+and+the+analysis+of+intercellular+signaling.">Wing vein patterning in Drosophila and the analysis of intercellular signaling.</a> Annu Rev Cell Dev Biol. 23, 293-319 (2007).</li><li>Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drawing+lines+in+the+Drosophila+wing:+initiation+of+wing+vein+development.">Drawing lines in the Drosophila wing: initiation of wing vein development.</a> Curr Opin Genet Dev. 10 (4), 393-398 (2000).</li><li>De Celis, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+formation+in+the+Drosophila+wing:+The+development+of+the+veins.">Pattern formation in the Drosophila wing: The development of the veins.</a> Bioessays. 25 (5), 443-451 (2003).</li><li>Classen, A. K., Aigouy, B., Giangrande, A., Eaton, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Drosophila+pupal+wing+morphogenesis.">Imaging Drosophila pupal wing morphogenesis.</a> Methods Mol Biol. 420, 265-275 (2008).</li><li>Bolatto, C., 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=Spatial+and+temporal+distribution+of+Patched-related+protein+in+the+Drosophila+embryo.">Spatial and temporal distribution of Patched-related protein in the Drosophila embryo.</a> Gene Expr Patterns. 19 (1-2), 120-128 (2015).</li><li>Zuniga, 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=Genes+encoding+novel+secreted+and+transmembrane+proteins+are+temporally+and+spatially+regulated+during+Drosophila+melanogaster+embryogenesis.">Genes encoding novel secreted and transmembrane proteins are temporally and spatially regulated during Drosophila melanogaster embryogenesis.</a> BMC Biol. 7, 61 (2009).</li></ol>

该视频中所描述的解剖方法可用于为许多类型的技术 (如,免疫荧光法, cDNA 合成, PCR 检测) 制备高质量的果蝇蛹翅的样品. 体外翼发育)。根据这种方法, 科学家们使用了 24-30 h 型 APF。然而, 在解剖年轻或年长的蛹的关键步骤中, 不应有任何障碍。可能必须作出改变, 以适应较小的阶段蛹。
该协议可以进行解剖一个蛹或几个蛹在同一时间。这有助于加快过程, 使...

果蝇机翼是从机翼形象盘中开发的。在胚胎发育过程中, 这些翼盘的原始性被放在一边, 作为20-30 形象细胞的小基团, 从胚胎上皮 invaginate。当幼虫生长到第三个龄期时, 有丝分裂发生在形象细胞在特定的特征时间提高其数量 (约50000细胞), 形成翼盘1,2,3, 4。当细胞增殖时, 它们会形成管状上皮, 形成 self-folding 的致密螺旋。在蛹期间, 圆盘上皮望远镜形成二层翼和纵脉开始作为他们之间的空白, 在翼发展期间发生两次5,6。
静脉的排列总是有相同的图案;能够很容易地识别血管形成中的每一个变化, 使得突变非常明显 (例如缺失或额外的静脉, 静脉位置的变化,等)。有趣的是, 表型变化通常是已知或新奇的信号传导通路的突变的证据, 它们与机翼的发育有关。在确定位置和维持静脉和交错区域中扮演角色的一个途径是刺猬 (Hh) 路径6,7,8。
考虑到蛹机翼作为一个模型系统的重要性, 获得高质量的样品是很重要的。过去, 科学家们已经发布了解剖果蝇翅膀的协议, 但并没有给出合适的指南来获得可行的样本。没有研究员的教导你不可能清楚地形象化过程。在这些交替的解剖方法中, 蛹被一分为二, 将机翼与周围的组织隔开, 并反复洗涤以除去碎片。这种方法声称离开机翼的污染, 但由于以往的经验的过程是较慢的, 有更大的机会破坏脆弱的机翼结构9。
这里描述的程序是根据需求找到一种快速和有效的方法获得蛹翼样本, 适合检测特定的蛋白质或抄本使用免疫协议或聚合酶链反应 (PCR) 化验。为了说明这一点, 在蛹翼样本中检测到了已修补的 (Ptc 或 Hh 通路的典范受体) 的表达和定位, 提供了包含相关细节的协议, 以成功地完成 程序.

<table border="0" cellpadding="0" cellspacing="0" width="809">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:349px;">Stereomicroscope with cool light illumination</td>
			<td style="width:175px;">Nikon&#160;</td>
			<td style="width:119px;">SMZ1000</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rocking platform</td>
			<td>Biometra (WT 16)</td>
			<td>042-500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope slides</td>
			<td>StarFrost</td>
			<td>8037/1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cover glasses (24 x 32 mm)</td>
			<td>Deltalab</td>
			<td>D102432</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps</td>
			<td>F.S.T</td>
			<td>11252-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors</td>
			<td>Lawton</td>
			<td>63-1400</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Multi-well plastic dish</td>
			<td>Thermo Scientific</td>
			<td>144444</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">p100 or p200 pipette</td>
			<td>Finnpipette (Labsystems)</td>
			<td>9400130</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1X PBS (Dulbecco)</td>
			<td>Gibco</td>
			<td>21600-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4% paraformaldehyde&#160;</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BSA</td>
			<td>Bio Basic INC.</td>
			<td>9048-46-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>Mallinckrodt</td>
			<td>5092</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris</td>
			<td>Amresco</td>
			<td>497</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TRIzol Reagent</td>
			<td>Ambion</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DEPC treated water</td>
			<td>MO.BIO</td>
			<td>17011-200</td>
			<td></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:349px;">Alexa Fluor 546 goat anti-mouse IgG (H+L), 2 mg/mL 
			(working dilution 1/800)</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">A11030</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="153">
			<td height="153" style="height:153px;width:349px;">Monoclonal anti-Ptc (Apa-1) 54 &#956;g/mL 
			(working dilution 1/100)</td>
			<td style="width:175px;">Developmental Studies Hybridoma Bank (DSHB)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">By Isabel Gerrero from DSHB under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.&#160;</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:349px;">DAPI, 1 mg/mL 
			(working dilution 1/5000)</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">62248</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="58">
			<td height="58" style="height:58px;width:349px;">Phalloidin conjugated to Alexa 488, 300 U/1.5 mL methanol 
			(1/600 working dilution)</td>
			<td style="width:175px;">Invitrogen</td>
			<td style="width:119px;">A12379</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:349px;">Canton S (fly stock)</td>
			<td style="width:175px;">Bloomington Drosophila Stock Center&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

a rapid and efficient method to dissect pupal wings of drosophila suitable for immunodetections or pcr assays

在果蝇的机翼发育是研究组织层形态发生的理想模型。这些附属物从胚胎发育过程中形成的一组叫做翼形象的细胞发展而来。在幼体阶段, 形象椎间盘生长, 增加细胞数量, 形成 monolayered 上皮结构。在蛹的情况下, 形象盘芽和折叠成双层沿一条线, 成为未来的翼缘。在这一过程中, 纵 primodia 脉在翼的准背和腹面上起源于静脉细胞。在蛹阶段, 每个表面的静脉细胞的条纹, 以产生致密管;与此同时, cross-veins 开始形成。
在适当的分子标记的帮助下, 它是可能的确定主要元素构成翼在它的发展期间。由于这个原因, 能够准确地检测到该结构中的转录或蛋白质, 对于研究它们的丰度和定位与机翼的发展过程是至关重要的。
这里介绍的过程集中在操纵蛹翼, 提供详细的说明如何解剖机翼在蛹阶段。蛹组织的解剖比它们在第三龄幼虫中的表现更难。这就是为什么这种方法的发展, 以获得快速和高效的高质量样品。有关如何免疫和安装这些机翼样品, 让蛋白质或细胞成分可视化的细节, 在协议中提供。由于缺乏专业知识, 可以在短时间内收集8-10 高品质的蛹机翼。

该协议提供了一个简单的方法获得蛹翼样本, 保留了熟悉的机翼形态。从这些准备工作中, 可以获得一堆可能被投射为2维或3维的图像, 以研究在机翼分化过程中蛋白质的分布和/或丰富度。类似的协议 (见2.9 的说明) 可以用来收集大蛹翼样本 (涉及50-80 蛹翼) 合成互补脱氧核糖核酸 (cDNA), 用于 PCR 反应的模板。
<img alt="Figure 1" class="...

Watch this Scientific Journal Video about A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays at JoVE.com

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

在果蝇组织中准确检测转录和蛋白质的能力对于研究它们与发展过程有关的丰度和定位是至关重要的。这里是一个简单的过程解剖蛹翅膀的描述。这些翅膀可以作为样品在一些技术 (免疫组织化学, PCR 检测,等)。

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 ...

Research

JoVE Journal

Developmental Biology

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

Live-imaging of the Drosophila Pupal Eye

Live-imaging of the Drosophila Pupal Eye

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

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

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

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