Name: a rapid and efficient method to dissect pupal wings of drosophila suitable for immunodetections or pcr assays
Uploaded: 2017-12-30T14:00:00.000+00:00
Duration: 7 min 11 s
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标志的, 被认为是 0 h APF。蛹是一个不动的白色幼虫, 有一个长方形, 圆形, 突出前气孔。</li>
</ol>
2天
2. 转移、解剖和固定。
<ol>
	<li>将蛹 (2 小时前的开发时间完成) 与一支画笔, 到显微镜幻灯片与双面胶带坚持它。使蛹形成一排背侧和头端朝向同一方向。</li>
	<li>将显微镜幻灯片返回到恒定温度, 并允许蛹完成适当的发育时间 (即24-30 h APF)。 
	注意:与双面胶带的显微镜滑动将是一个暂时的解剖平台, 以消除蛹的情况下 (见下文)。显微镜滑动 (瓶外) 的时间推移有助于干燥蛹的情况下, 使其容易破碎的钳子。</li>
	<li>用镊子将盖从蛹箱中取出。</li>
	<li>用钳子 (如视频中所示) 打破这一情况, 沿着蛹的一侧到它的尾端线。有了适当的光照, 就可以检测蛹的内部形状, 使其在蛹翼的铰链上方有一个明显的空间。这个空间作为一个指南, 以执行开放, 而不损害蛹。</li>
	<li>删除部分的情况下, 拾起他们在打开的边界, 并携带他们到对面的双面胶带将举行他们。在解剖的最后, 蛹几乎是 "赤裸", 只有一小部分的情况下将覆盖后端。 注: 在上尾端留下少量的情况是为了确保从机箱上平滑地释放到带有双面胶带的滑块上。这一行动背后的原因是, 如果你删除太多的情况下, 你很大的风险损害蛹。另一方面, 如果你删除太少, 蛹将不容易来摆脱的情况。</li>
	<li>用镊子把箱子的其余部分往下推。这一运动将提高蛹的前端, 使头部和胸部保持在一个优先地位, 以坚持和转移在随后的步骤。</li>
	<li>用双面胶带将新的幻灯片翻转到蛹或蛹排。我们建议采取这种方法, 观察运动的帮助下, 显微镜防止蛹的破坏。</li>
	<li>稍微按压一下, 使蛹粘在胶带上, 然后轻轻地滑动, 以便从其余的病例中取出蛹。倒置显微镜幻灯片: 蛹将被卡在背侧的水平, 他们的头部/胸部区域。</li>
	<li>覆盖所有的裸蛹与4% 甲醛和等待10分钟。根据我们的经验, 这种固定的时间的推移有助于改变角质层的一致性, 使其牢固, 不粘, 易于处理。 
	注意:为了执行核糖核酸 (RNA) 提取, 替代甲醛的磷酸盐缓冲盐水 (PBS) 制成的核糖核酸自由水和删除的蛹翅膀作出削减 (借助剪刀) 的铰链点附近的每个附属物。用镊子将蛹翼转移到离心管上, 用1毫升的硫氰酸胍和苯酚试剂。收集了总共50-80 个机翼后, 将离心管存储在-80 ° c, 直到执行 RNA 提取10,11。</li>
	<li>在机翼的铰链区域或靠近它的地方做一个小切口。使定影液到达翼上皮。在机翼上使用吸管 (p200) 冲洗定影液。当冲洗取代被污染的执着与新。冲洗后, 将机翼固定在定影液中5分钟。 
	注意:虽然这种机动有助于清除大部分的污染组织, 一些血和脂肪滴可能仍然被困在背部和腹侧上皮层之间的机翼 (见图 1A)。</li>
	<li>撕裂与钳子从角质层边界 (如在视频中所示) 扩大孔, 并使释放的翼上皮从角质层囊。一旦机翼组织被释放, 留在甲醛溶液中完成30分钟的固定。</li>
	<li>将固定翼转移到一个满是 PBST (海卫 X-100 0.05%) 的4溢的塑料碟中。在4° c 过夜。</li>
</ol>
3天
3. 主要抗体的培养。
<ol>
	<li>Permeabilize 的组织孵化蛹翼在 PBST (海卫 X-100 0.2%) 为15分钟使用摇摆平台, 以促进温和的液体介质运动。 
	注意:在安装这些示例之前, 应使用摇摆平台执行这些步骤。</li>
	<li>使用 PBSTA (BSA 3%, 海卫 X-100 0.1%) 为1小时阻止样品。</li>
	<li>使用相同的缓冲液来稀释主抗体 (小鼠抗 Ptc, 1:100), 在4° c 的夜间孵育蛹翅。</li>
</ol>
4天
4. 二级抗体培养。
<ol>
	<li>洗涤样品 4 x 10 min 每个与 PBST (海卫 X-100 0.05%)</li>
	<li>孵育与二次抗体 (例如, anti-mouse 共轭到荧光) 稀释到适当的集中在 PBSTA (BSA 3%, 海卫 X-100 0.1%) 在室温下在黑暗中, 为 2 h。 
	注意:如果合适, 使用 4 ", 6-diamidino-2-吲 (DAPI) 和/或笔 counterstain 的组织。将这些探针与二次抗体结合, 考虑到荧光的发射波长, 以防止信号重叠。</li>
	<li>洗涤样品 4 x 10 min 每个与 PBST (海卫 X-100 0.05%)</li>
</ol>
5. 安装
<ol>
	<li>在显微镜上放置4点透明指甲油, 准备一个样品幻灯片, 就好像它们是正方形的顶点一样。</li></ol>他们将是片将休息, 帮助避免粉碎组织的点。
	<li>放置30µL 的安装介质 (甘油/三-HCl 80%) 在广场中心的点, 用指甲油, 而不触及任何角落 (点)。取一个带有黄色裁剪尖端的吸管, 装上一些安装介质, 然后用几个蛹的翅膀画出。</li>
	<li>将蛹的机翼转移到显微镜滑轨上的安装介质的点上。在尖端的帮助下, 传播安装媒体和下沉蛹的翅膀, 以防止他们漂浮。当盖上玻璃被放置时, 如果机翼保持平坦, 组织的形态将保持不变。在放置盖子玻璃之前, 除去气泡。</li>
	<li>将片放在样品上, 试图防止气泡的产生。这一运动可以借助镊子来完成。</li>

<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>

作者没有什么可透露的。

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><tbody><tr><td>Stereomicroscope with cool light illumination</td><td>Nikon&amp;nbsp;</td><td>SMZ1000</td><td></td></tr><tr><td>Rocking platform</td><td>Biometra (WT 16)</td><td>042-500</td><td></td></tr><tr><td>Microscope slides</td><td>StarFrost</td><td>8037/1</td><td></td></tr><tr><td>Cover glasses (24 x 32 mm)</td><td>Deltalab</td><td>D102432</td><td></td></tr><tr><td>Forceps</td><td>F.S.T</td><td>11252-00</td><td></td></tr><tr><td>Scissors</td><td>Lawton</td><td>63-1400</td><td></td></tr><tr><td>Multi-well plastic dish</td><td>Thermo Scientific</td><td>144444</td><td></td></tr><tr><td>p100 or p200 pipette</td><td>Finnpipette (Labsystems)</td><td>9400130</td><td></td></tr><tr><td>1X PBS (Dulbecco)</td><td>Gibco</td><td>21600-010</td><td></td></tr><tr><td>4% paraformaldehyde&amp;nbsp;</td><td>Sigma</td><td>158127</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>BSA</td><td>Bio Basic INC.</td><td>9048-46-8</td><td></td></tr><tr><td>Glycerol</td><td>Mallinckrodt</td><td>5092</td><td></td></tr><tr><td>Tris</td><td>Amresco</td><td>497</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Ambion</td><td>15596026</td><td></td></tr><tr><td>DEPC treated water</td><td>MO.BIO</td><td>17011-200</td><td></td></tr><tr><td>Alexa Fluor 546 goat anti-mouse IgG (H+L), 2 mg/mL&lt;br/&gt; (working dilution 1/800)</td><td>Invitrogen</td><td>A11030</td><td></td></tr><tr><td>Monoclonal anti-Ptc (Apa-1) 54 &amp;mu;g/mL&lt;br/&gt; (working dilution 1/100)</td><td>Developmental Studies Hybridoma Bank (DSHB)</td><td></td><td>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.&amp;nbsp;</td></tr><tr><td>DAPI, 1 mg/mL&lt;br/&gt; (working dilution 1/5000)</td><td>Invitrogen</td><td>62248</td><td></td></tr><tr><td>Phalloidin conjugated to Alexa 488, 300 U/1.5 mL methanol&lt;br/&gt; (1/600 working dilution)</td><td>Invitrogen</td><td>A12379</td><td></td></tr><tr><td>Canton S (fly stock)</td><td>Bloomington Drosophila Stock Center&amp;nbsp;</td><td></td><td></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 ...

a-rapid-efficient-method-to-dissect-pupal-wings-drosophila-suitable

Universidad de Cartagena UDEC

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JoVE Journal

Developmental Biology

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

8.0K 次观看.  Laboratorio de Biología del Desarrollo. 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)。

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

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and mechanobiology, which all influence muscle development and myofibrillogenesis. Omics data, such as those generated by mass spectrometry or deep sequencing, can provide important mechanistic insights into these biological processes. For such approaches, it is beneficial to analyze tissue-specific samples to increase both selectivity and specificity of the omics fingerprints. Here we present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched muscle samples for omics applications. We first describe how to dissect flight muscles at early pupal stages (&#60;48 h after puparium formation [APF]), when the muscles are discernable by green fluorescent protein (GFP) labeling. We then describe how to dissect muscles from late pupae (&#62;48 h APF) or adults, when muscles are distinguishable under a dissecting microscope. The accompanying video protocol will make these technically demanding dissections more widely accessible to the muscle and Drosophila research communities. For RNA applications, we assay the quantity and quality of RNA that can be isolated at different time points and with different approaches. We further show that Bruno1 (Bru1) is necessary for a temporal shift in myosin heavy chain (Mhc) splicing, demonstrating that dissected muscles can be used for mRNA-Seq, mass spectrometry, and reverse transcription polymerase chain reaction (RT-PCR) applications. This dissection protocol will help promote tissue-specific omics analyses and can be generally applied to study multiple biological aspects of myogenesis.

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study transcriptional regulation, alternative splicing, metabolism, and mechanobiology. We present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched samples ideal for proteomics and deep-sequencing. These samples can offer important mechanistic insights into diverse aspects of muscle development.

解剖果蝇黑色素组飞行肌肉为奥米克斯方法

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and ...

科研

遗传学

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

神经科学

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

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 ...

发育生物学

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and behavioral evolution. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons. In this protocol, we describe the dissection of developing peripheral olfactory tissue of the adult Drosophila species. We first describe how to stage and age Drosophila larvae, followed by the dissection of the antennal disc from early pupal stages, followed by the dissection of the antennae from mid-pupal stages and adults. We also show methods where preparations can be utilized in molecular techniques, such as the RNA extraction for qRT-PCR, RNAseq, or immunohistochemistry. These methods can also be applied to other Drosophila species after species-specific pupal development times are determined, and respective stages are calculated for appropriate aging.

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Here, we present a protocol to stage and dissect developing olfactory tissue from Drosophila species. The dissected tissue can later be used for molecular analyses, such as quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) or RNA sequencing (RNAseq), as well as in vivo analyses such as immunohistochemistry or in situ hybridization.

果蝇分子和免疫组化分析制备周围嗅组织的研制

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and ...

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

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

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

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

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 ...

Imaging Dpp Release from a Drosophila Wing Disc

The transforming Growth Factor-beta (TGF-&#946;) superfamily is essential for early embryonic patterning and development of adult structures in multicellular organisms. The TGF-&#946; superfamily includes TGF-&#946;, bone morphogenetic protein (BMPs), Activins, Growth and Differentiation Factors, and Nodals. It has long been known that the amount of ligand exposed to cells is important for its effects. It was thought that long-range concentration gradients set up embryonic pattern. However, recently it has become clear that the timing of exposure to these ligands is also important for their downstream transcriptional consequences. A TGF-&#946; superfamily ligand cannot have a developmental consequence until it is released from the cell in which it was produced. Until recently, it was difficult to determine when these ligands were released from cells. Here we show how to measure the release of a Drosophila BMP called Decapentaplegic (Dpp) from the cells of the wing primordium or wing disc. This method could be modified for other systems or signaling ligands.

Imaging Dpp Release from a Drosophila Wing Disc

The timing of exposure to ligands may impact their developmental consequences. Here we show how to image release of a Drosophila bone morphogenetic protein (BMP) called Dpp from cells of the wing disc.

成像 Dpp 释放从果蝇翼光盘

The transforming Growth Factor-beta (TGF-&#946;) superfamily is essential for early embryonic patterning and development of adult structures in ...

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.

果蝇蛹腹部免疫

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

生物学

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses&#160;and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle &#8211; the Drosophila pupa &#8211; offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

果蝇蛹中炎症细胞动力学的长期体内追踪

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, ...

免疫与感染

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent adult integument. Their immobility and transparency make them ideal for in vivo live imaging experiments. Many studies have focused on the dorsal epithelial monolayer of the pupal&#160;notum because of its accessibility and relatively large size. In addition to the studies of epithelial mechanics and development, the notum has been an ideal tissue to study wound healing. After an injury, the entire epithelial repair process can be captured by live imaging over 6-12 h. Despite the popularity of the notum for live imaging, very few published studies have utilized fixed notum samples. Fixation and staining are common approaches for nearly all other Drosophila tissues, taking advantage of the large repertoire of simple cellular stains and antibodies. However, the pupal notum is fragile and prone to curling and distortion after removal from the body, making it challenging to complement live imaging. This protocol offers a straightforward method for fixing and staining the pupal notum, both intact and after laser-wounding. With this technique, the ventral side of the pupa is glued down to a coverslip to immobilize the pupa, and the notum is carefully removed, fixed, and stained. The notum epithelium is mounted on a slide or between two coverslips to facilitate imaging from the tissue&#39;s dorsal or ventral side.

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The present protocol details the preparation and visualization of fixed tissue of the Drosophila pupal notum. It can be used for either intact or wounded tissue, and the original architecture of the tissue is preserved. The procedures for dissecting, fixing, and staining are all described in this article.

解剖、固定和可视化 果蝇 蛹

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent ...

Drosophila Larva Imaginal Disc Dissection: A Method to Observe Developing Epithelia

Source: Morimoto, K. and Tamori, Y. <a href="https://jove.unicartagenaproxy.elogim.com/video/55901/" target="_blank">Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia</a>. J. Vis. Exp. (2017).
When staining Drosophila imaginal discs, often a rough dissection is performed where the discs are exposed but remain attached to the body. Once stained, the discs of interest are isolated and mounted. The example protocol demonstrates a procedure for wing imaginal disc dissection and mounting.

果蝇幼虫假想盘清扫术:一种观察发育中的上皮细胞的方法

Source: Morimoto, K. and Tamori, Y. Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia. J. Vis. Exp. (2017).
When staining Drosophila ...

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

摘要

探索更多视频

此视频中的章节

解剖果蝇黑色素组飞行肌肉为奥米克斯方法

可视化本体感受器

果蝇蛹卵巢的解剖与染色

果蝇分子和免疫组化分析制备周围嗅组织的研制

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

成像 Dpp 释放从果蝇翼光盘

果蝇蛹腹部免疫

果蝇蛹中炎症细胞动力学的长期体内追踪

解剖、固定和可视化果蝇蛹

果蝇幼虫假想盘清扫术:一种观察发育中的上皮细胞的方法

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

摘要

探索更多视频

此视频中的章节

解剖果蝇黑色素组飞行肌肉为奥米克斯方法

可视化本体感受器

果蝇蛹卵巢的解剖与染色

果蝇分子和免疫组化分析制备周围嗅组织的研制

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

成像 Dpp 释放从果蝇翼光盘

果蝇蛹腹部免疫

果蝇蛹中炎症细胞动力学的长期体内追踪

解剖、固定和可视化 果蝇 蛹

果蝇幼虫假想盘清扫术:一种观察发育中的上皮细胞的方法

解剖、固定和可视化果蝇蛹