作者感谢SI伯恩斯坦（圣迭戈州立大学）批判性阅读和有益的建议，关于这个手稿的准备。这项工作是由美国国立卫生研究院拨款支持SI伯恩斯坦，SDSU的，和R.博德默，BIMR;和一个博士后奖学金从西方国家的美国心脏协会联盟G. Vogler和A. Cammarato。

在您开始之前<ol><li>准备了以下解决方案： <ol class="lalpha"><li>放宽缓冲区（人工果蝇血淋巴（ADH）的（见“可视化在果蝇中跳动的心脏”），其中包含10 mM的EGTA） </li><li>固定液（4％甲醛1X PBS） </li><li> PBSTx（用含0.1％TRITON - X - 100的） </li><li>适当稀释的小学和物种特异性荧光标记PBSTx中学的抗体</li></ol></li><li>解剖果蝇暴露心脏管（成人苍蝇和/或幼虫） <ol class="lalpha"><li>在“可视化在果蝇的心脏跳动 ”半完整的果蝇心脏准备协议，或</li><li> 果蝇幼虫NMJ夹层协议 1以下修改：使用含氧在幼虫夹层ADH和取代的腹侧中线后针稍微。幼虫沿腹中线被切断。不要删除任何组织内固定前。 </li></ol></li></ol>荧光染色<ol><li>检查所有心中有节奏地跳动含氧ADH的。迅速取代放宽缓冲液的乙醇脱氢酶。检查每个心管，以确保收缩有抑制作用。 </li><li>修复取代放宽缓冲固定液的心。在室温下孵育20分钟，轻轻摇动。 （幼虫准备晃动是没有必要在任何步骤，并可能被视为有损心脏组织的完整性）。 </li><li>洗净标本与PBSTx 10分钟，在室温下不断晃动三次。 </li><li>对于成年人来说，精心修剪等，剩下的就是更多的椭圆形，少圆润的腹部角质层的腹侧边缘。此外，确保与腹部和胸部之间的单切，仔细和清楚分开的两个身体细分和心前区的最小的损害。幼虫的心，用细镊子小心取出脂肪体。去除脂肪，必须执行非常谨慎，因为幼虫的心，尤其是脆弱的，并已很少从其他肌肉或结缔组织的支持。不要取出气管分支，因为这可能会损害心脏。 </li><li>的边缘修剪背腹部角质层的地区转移，避免接触到一个96口井，包含50-100μl稀释的抗体在PBSTx的孔板，位于市中心的心脏管。将不超过12％以及标本。在室温下孵育2小时，不断搅拌。孵化可能一蹴而就，在4 ° C。 </li><li>删除第一抗体的解决方案。 10分钟在室温下用100μLPBSTx不断晃动的心洗净三次。 </li><li>继拆除最后的洗涤缓冲液，添加在PBSTx 100μL二次抗体，辅以Alexa594 -鬼笔环肽（1:1000）。在室温下持续摇晃一小时孵育。保持样本覆盖，防止荧光团的漂白。 </li><li>二次孵化之后，心中洗为10分钟三次在室温下持续晃动100μLPBSTx。保持整个洗涤步骤所涉及的样本。 </li><li>拆除TRITON - X - 100，100μL的PBS冲洗心中最后的10分钟时间。标本可以保存在4 ° C安装前几天在黑暗中。 </li></ol>成人心脏安装<ol><li>坚持两个18 × 18毫米的盖玻片，显微镜幻灯片与10μLVectashield安装介质。在盖玻片应间隔〜10-15毫米。安装在两个盖玻片之间的媒介放置一个小的第三个下拉。 </li><li>将在第三盖玻片中心20μLVectashield。 </li><li> PBS洗涤液，小心地取出心中的极端边缘的角质​​层，并轻轻地放到他们心端安装介质下降第三盖玻片。盖玻片放在心中不超过5％下降。 </li><li>在显微镜下检查，以确保所有的心都朝下。 </li><li>小心反转盖玻片的心，并迅速在幻灯片上放置含有盖玻片对这样Vectashield液滴之间的盖玻片对心脏管保险丝的液滴。 “桥梁”，应成立由盖玻片。盖玻片，在显微镜幻灯片之间的心将暂停。 </li><li>在显微镜下检查，以确保现在的心是朝上。 </li><li>指甲油的边缘固定在盖玻片。 </li><li>现在随时可以通过荧光或共聚焦显微镜成像的心。 </li></ol><ol><li>将载玻片上滴（约20μL）Vectashield。 </li><li>小心地转移到Vectashield和东方背侧下来两个幼虫标本使用钨针。 </li><li>单独拖动标本的安装介质和调整每个并行。 </li><li>将试样两侧盖玻片。使用镊子，放置在顶部的第三盖玻片，首先由后盖玻片一侧铺设降下来前盖玻片，从而形成了一个桥梁。毛细管的力量将导致Vectashield从后到前这有助于正确对齐的幼虫的心流。 </li><li>固定盖玻片，在其边缘，指甲油，并认真填写20-30μLVectashield的盖玻片之间的空间。 </li><li>指甲油和存储密封在4 ° C，或长期储存在-20 ° C </li></ol>代表性的成果正确执行时，背船只的所有组件和相关的组织应保持不变，很容易地可视化。背景荧光应该不大。对于成年人，腹侧纵肌层污渍非常好，并产生了大量的信号（图1和图2）。底层圆形心肌然而，往往不产生激烈的一个信号覆腹层。前的“锥形腔”成人心脏的心肌细胞含有大量的收缩材料，因此，本地区最强大的心脏管的其余部分相出现。幼虫的心，显示了一个螺旋式上升的肌原纤维的安排，类似成人收缩的心肌细胞（图3）。 <img src="/files/ftp_upload/1423/1423fig1atmb.jpg" alt="图1a" border="0" /> <img src="/files/ftp_upload/1423/1423fig1btmb.jpg" border="0" alt="图1b" /> 图1：顶。一种荧光显微照片显示的整个成年果蝇心脏管，表达肌球蛋白GFP。形象是一个蔡司成像仪配备了一个Apotome滑动模块的Z1的荧光显微镜。肌球蛋白GFP显示为绿色，肌动蛋白是与AlexaFluor ® 594鬼笔环肽（红色）和α-辅肌动蛋白染色标记的抗-α-辅肌动蛋白抗体（蓝色）。注意：制备过程中允许恢复心脏样本保存完好的结构。 CC =锥形腔; AM = alary肌肉，V =内部阀; VLL的腹侧纵肌层。底部。通过第三腹部下方呈现不断上升的肌原纤维的收缩心肌安排的腹侧纵层的核心部分CC成人心脏管区域。请<a href="https://www.jove.com/files/ftp_upload/1423/1423fig1a.jpg">点击这里</a>图1a为一个更大的版本，并<a href="https://www.jove.com/files/ftp_upload/1423/1423fig1b.jpg">在这里</a>图1b为一个更大的版本<img border="0" src="/files/ftp_upload/1423/1423fig2tmb.jpg" alt="图2" /> 图2：AlexaFluor ® 594鬼笔环肽（红色）和反Pericardin（IV型胶原）（绿色）抗体染色成人心脏的前部的代表共聚焦栈。 Pericardin是与沿腹面的心，有可能从腹侧的肌肉层的纵向为主的肌原纤维的。请<a href="https://www.jove.com/files/ftp_upload/1423/1423fig2.jpg">点击这里</a>为图2的较大版本。 <img border="0" src="/files/ftp_upload/1423/1423fig3tmb.jpg" alt="图3" /> 图3：一个分部了第三阶段的果蝇幼虫心脏A7的荧光显微镜。形象是一个蔡司成像仪配备了一个Apotome滑动模块的Z1的荧光显微镜。肌动蛋白是标记AlexaFluor ® 594鬼笔环肽（绿色）和α-辅肌动蛋白是染色与抗-α-辅肌动蛋白抗体（红色）。请<a href="https://www.jove.com/files/ftp_upload/1423/1423fig3.jpg">点击这里</a>为图3的较大版本。

<ol>
	<li>Brent, J. R., Werner, K. M., McCabe, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+larval+NMJ+dissection.">Drosophila larval NMJ dissection.</a> J Vis Exp. , (2009).</li><li>Molina, M. R., Cripps, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ostia,+the+inflow+tracts+of+the+Drosophila+heart,+develop+from+a+genetically+distinct+subset+of+cardial+cells.">Ostia, the inflow tracts of the Drosophila heart, develop from a genetically distinct subset of cardial cells.</a> Mech Dev. 1, 51-59 (2001).</li><li>Dulcis, D., Levine, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamatergic+innervation+of+the+heart+initiates+retrograde+contractions+in+adult+Drosophila+melanogaster.">Glutamatergic innervation of the heart initiates retrograde contractions in adult Drosophila melanogaster.</a> J Neurosci. 2, 271-280 (2005).</li><li>Zeitouni, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling+pathways+involved+in+adult+heart+formation+revealed+by+gene+expression+profiling+in+Drosophila.">Signalling pathways involved in adult heart formation revealed by gene expression profiling in Drosophila.</a> PLoS Genet. 10, 1907-1921 (2007).</li><li>Robertson, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metamorphosis+of+Drosophila+melanogaster,+including+an+accurately+timed+account+of+the+principal+morphological+change.">The metamorphosis of Drosophila melanogaster, including an accurately timed account of the principal morphological change.</a> J. Morphol. 2, 351-399 (1936).</li><li>Miller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The internal anatomy and histology of the imago of Drosophila melanogaster. , (1950).</li><li>Rizki, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circulatory+system+and+associated+cells+and+tissues.">The circulatory system and associated cells and tissues.</a> , (1978).</li><li>Cammarato, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin+transducer+mutations+differentially+affect+motor+function,+myofibril+structure,+and+the+performance+of+skeletal+and+cardiac+muscles.">Myosin transducer mutations differentially affect motor function, myofibril structure, and the performance of skeletal and cardiac muscles.</a> Mol Biol Cell. 2, 553-562 (2008).</li><li>Taghli-Lamallem, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dystrophin+deficiency+in+Drosophila+reduces+lifespan+and+causes+a+dilated+cardiomyopathy+phenotype.">Dystrophin deficiency in Drosophila reduces lifespan and causes a dilated cardiomyopathy phenotype.</a> Aging Cell. 2, 237-249 (2008).</li><li>Kelso, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+database+documenting+a+GFP+protein-trap+insertion+screen+in+Drosophila+melanogaster.">a database documenting a GFP protein-trap insertion screen in Drosophila melanogaster.</a> Nucleic Acids Res. 32, D418-D420 (2004).</li><li>Dietzl, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+transgenic+RNAi+library+for+conditional+gene+inactivation+in+Drosophila.">A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.</a> Nature. 7150, 151-156 (2007).</li><li>Mery, A. <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+muscle+LIM+protein,+Mlp84B,+is+essential+for+cardiac+function.">The Drosophila muscle LIM protein, Mlp84B, is essential for cardiac function.</a> J Exp Biol. 211, 15-23 (2008).</li></ol>

在这里，我们提出了一个协议， 准备和染色果蝇背血管和组织通过荧光或共聚焦显微镜成像。我们提供一个简洁的细化和有效的染色， 允许在幼虫和成虫果蝇心管原位成像解决通常在我们的实验室工作的步骤帐户。其他缩写形式2，3，4所描述的类似的方法。 实施染色过程时，重要的额外细节需要考虑。例如，与其他生物材料中发现，相当<...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Ethylene glycol-bis (2-amino-ethylether) -N,N,N&#x2019;,N&#x2019;-tetra-acetic acid (EGTA)</td>
<td>Reagent</td>
<td>Sigma-Aldrich&reg;</td>
<td>E4378</td>
<td>&nbsp;</td>
<tr>
<td>Formaldehyde, 10%, methanol free, Ultra Pure</td>
<td>Reagent</td>
<td>Polysciences Inc.</td>
<td>50-00-0</td>
<td>&nbsp;</td>
<tr>
<td>Triton-X-100</td>
<td>Reagent</td>
<td>Sigma-Aldrich&reg;</td>
<td>9002-93-1</td>
<td>&nbsp;</td>
<tr>
<td>Alexa Fluor&reg; 594 phalloidin</td>
<td>Reagent</td>
<td>Invitrogen&trade;</td>
<td>A12381</td>
<td>&nbsp;</td>
<tr>
<td>Vectashield&reg; Mounting Medium for Fluorescence with DAPI</td>
<td>Reagent</td>
<td>Vector Laboratories, Inc.</td>
<td>H-1200</td>
<td>&nbsp;</td>
<tr>
<td>Tungsten pins</td>
<td>Reagent</td>
<td>Fine Science Tools</td>
<td>26002-10</td>
<td>&nbsp;</td>
<tr>
<td>Pin holder</td>
<td>Reagent</td>
<td>Fine Science Tools</td>
<td>26018-17</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fluorescent labeling of drosophila heart structures

“<em&gt;果蝇</em&gt;背船只，或心脏，是一个收缩的心肌，心包细胞沿心墙，支持alary肌肉和在成人，腹纵肌细胞层两侧排列的单层组成的管状结构。收缩纤维的房子保守的肌肉细胞结构成分，包括肌原纤维和细胞骨架/ submembranous蛋白复合物，与同源的细胞外基质的组成部分相互作用束密密麻麻。在这里，我们描述了从解剖的幼虫和半完整的成人心中的一个固定的协议，特别是心肌元素的荧光标记<em&gt;果蝇</em&gt;。具体来说，我们表现出的肌节的F -肌动蛋白和α-辅肌动蛋白在幼虫心中的标签。此外，我们执行标签的F -肌动蛋白和α-辅肌动蛋白，肌球蛋白GFP表达成蝇和α-辅肌动蛋白和pericardin，IV型细胞外基质的胶原蛋白，在野生型成人的心。特别注意的是，以一个半完整的成人心中的安装策略，最大限度地减少了处理和优化的机会，为维护心管及相关组织的完整性。这些准备工作都适合通过荧光和共聚焦显微镜成像。总体而言，这个程序允许的心从一个强大的基因听话的模型系统的结构特点周密细致的分析。

Watch this Scientific Journal Video about Fluorescent Labeling of Drosophila Heart Structures at JoVE.com

Fluorescent Labeling of Drosophila Heart Structures

在这里，我们描述了从幼虫和成虫心管的不同元素的荧光标记的一个基本协议<em&gt;果蝇</em&gt;。这些标本非常适合通过荧光或共聚焦显微镜成像。这种技术允许一个强大的模式生物的心功能的详细的结构分析。

荧光标记的果蝇心结构

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

fluorescent-labeling-of-drosophila-heart-structures

Research

JoVE Journal

Biology

Fluorescent Labeling of Drosophila Heart Structures

13.6K 次观看.  San Diego State University. 在这里，我们描述了从幼虫和成虫心管的不同元素的荧光标记的一个基本协议果蝇。这些标本非常适合通过荧光或共聚焦显微镜成像。这种技术允许一个强大的模式生物的心功能的详细的结构分析。

视频: Fluorescent Labeling of Drosophila Heart Structures

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

科研

生物学

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

可视化的Live果蝇胶质细胞神经​​肌肉交界处，与荧光染料

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

可视化心脏跳动果蝇</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

在幼虫的监测心脏功能果蝇生理研究

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

果蝇聚乙烯南瓜抗体标记染色体的制备

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

对活细胞成像的SNAP - Tag融合蛋白的荧光标记的COS - 7

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

全山RNA荧光<em在原位</em&gt;杂交<em&gt;果蝇</em&gt;胚胎

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

果蝇的制备及心功能体内使用光学相干显微镜成像纵向（OCM）

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...