我们想感谢她的协助，在拍摄这段视频文章Amsale扎绳。这项工作是由CIHR，NSERC和CRC赠款JDL。

第1部分。圆角准备的成年果蝇腹部。 立即在下面的准备过程概述腹部oenocytes随后拆除（见议定书第2部分）。同样的程序也可以用于连接到内表面的角质层的其他组织，包括背血管（即心脏）和脂肪体的准备。 请注意，在视频文章oenocyte夹层技术是成年的野生型（广东）男飞，6天的年龄证明。相同的技术可以用于去除雌蝇oenocytes。腹部圆角准备大约需要5分钟来执行。 开始之前重要的是有准备的罚款几个清扫引脚（见下文第2部分）和一个夹层针（见第2部分）。市售清扫如坐​​针毡此协议往往过大，所以我们已在实验室中制造我们自己的。要创建解剖针，钨丝（0.005“）是第一个线程通过皮下注射针头的轴（27G，铝轮毂）。主题导线通过皮下注射针头的尖端，使其从集线器出现。压线凸出枢纽;防止拔出注射针丝下一步，切压等几个毫米的导线（约3 - 4mm的）从注射针的尖端突出的另一端线的传递。通过火焰的尖端暴露钨丝会激化到非常细点。一个一次性5毫升的塑料吸管时注射针枢纽楔形此清扫工具方便处理的行为。罚款清扫引脚类似使用方式。锐化一步后，抓紧与钳非常尖端的电线，以防止其移动和禁约3mm的尖端释放的小夹层针。护理，防止针被从遥远的把握镊子。 要圆角准备成年果蝇腹部： <ol><li>安全飞行夹层板（Sylgard;陶氏 - 康宁）置于胸部虽然罚款解剖针。 </li><li>删除飞的腿和翅膀。虽然卸下腿最后的设置，创建点前胸部符合腹部在角质层的开放。这种开放是作为一个接入点，沿腹部腹面切口后（见步骤5） </li><li>安全飞上腹部生殖器段第二解剖针。 </li><li>盖飞用冷冻肉盾牌和桑供应M3的昆虫培养基（Sigma公司）。用巴斯德吸管吸取删除任何被困的空气。 </li><li>使用镊子打开腹部，沿腹中线消除腿部生殖器段创建角质层开幕切口。删除从腹部的胆量和性腺。 </li><li>断绝角质层和气管连接到胸部腹部。剩下的唯一的组织连​​接到胸部腹部应心。这提供了一些稳定的腹部角质层，防止旋转或扭曲在下一步。 </li><li>针扁平的腹部角质层最前的角落。取出的胸部。拉角质层绷紧，针扁平的角质层后的角落。在生殖器段的脚可能需要重新定位，使角质层平放。 </li></ol>第2部分。 Oenocyte夹层腹部圆角准备公开腹部角质层和连接的组织，包括心脏，脂肪体，气管，体壁肌肉，表皮，和oenocytes的内部表面。核心在于沿背中线。脂肪体（不透明的组织），涵盖最内部的表皮表面。气管（白管状结构）从横向气门开口，延长，并广泛渗透到这些组织的分支机构。躺在这些组织下，色素oenocytes（琥珀色细胞）从中线外侧边缘的角质​​层在各分部后地区的辐射。 oenocytes在于直接下每个背片（背表皮板）的重水复疑无路鞣制的地区，可没有事先了解自己的位置难以确定。另一个腹侧位于oenocytes人口与sternites（腹侧表皮板），虽然有可能分离出这些细胞，这个视频协议只能证明背oenocytes清扫。在各腹节，脂肪体组织中的一个突出的叶在于前的oenocytes;脂肪体叶片较小的不太明显的谎言后的oenocytes。脂肪体组织中的突出叶往往涵盖了oenocytes的前面部分。背血管和脂肪体，必须先移除，以揭露的oenocytes。 重要的是要注意的oenocytes琥珀色素沉着，从他们的名字来源于（奥埃诺-从希腊 oînos，“酒”）1，明确区分这些细胞从周围组织的色素沉着的强度与年龄的增长苍蝇。此外，基底层ensheathes oenocytes 2,3，提供支持的细胞团，并防止成为在解剖分离的细胞。基底层还取消extraneously坚持组织，如脂肪体组织，在艾滋病，通过创建一个薄弱点，这些粘连，可以很容易地扰乱。 下文概述的程序提供了详细说明oenocytes去除腹部包膜。 oenocytes可以很容易地从腹部段2-5（男），和2-6（女）。解剖控股的角质层的角落引脚的第一个和最后一个腹段（段1，男性或女性7）oenocytes往往打乱，并因此避免。取消oenocytes大约需要10分钟来执行。 要隔离成人oenocytes： <ol><li>使用夹层针的气孔产生，切断气管树干。 </li><li>切断连接到内表面的角质层与解剖针取出脂肪的身体和心。要做到这一点，位置的夹层之间的脂肪体和oenocytes针点，并拖动从外侧边缘的角质​​层，通过中线，心脏下方移动，并持续到对面。进步从后到前，去除脂肪的身体组织，从每个连续段的角质层和心脏。如果进行仔细oenocytes保持角质层，和肥胖的身体和心可以删除整个。 </li><li>体壁肌肉表带oenocytes内表面的角质层。用解剖针断绝每个段的纵向体壁肌肉。要做到这一点，拖oenocyte链后，解剖针，轻轻提起他们从表面的角质层。剪彩像oenocyte股应该远离角质层，可铺设到一边，而其余oenocytes被删除。 </li><li>一旦从角质层，躺在夹层板，它可能是必要的清理关闭任何坚持从oenocytes脂肪的身体组织。这是只需将夹层之间的脂肪体组织和oenocytes针，针推下来，对夹层板和切断连接。 </li><li>通过简单地用解剖针戳他们收集oenocyte股。一旦坚持到oenocytes可以拉出整个培养基表面慢慢解剖针年底。解剖oenocytes可以直接放到适合RNA或蛋白质提取的细胞裂解液。请注意，从单一的男性飞oenocytes（2-5腹节）通常产量15 - 20ng的总RNA。 </li></ol>图例： <img src="/files/ftp_upload/2242/2242fig1.jpg" alt="图1" /> 图1。表达的去饱和酶基因，desat1和desatF D 的男性和女性oenocytes， 果蝇 。逆转录- PCR扩增，从cDNA desat1 和 desatF成年男性和女性oenocytes的所得。 desat1基因的表达在男性和女性oenocytes; desatF表达的是性二态，女性oenocytes表示。通讯desat1这些基因的表达差异，是为烃类化合物，常见的男性和女性 9的生产需要，参与的女性特有的烃类化合物10,11的生产 desatF。desatF已被证明是具体 ​​表现在女性oenocytes 原位杂交12。请注意，平均15 - 20ng总RNA是从个别飞解剖oenocytes获得。 RNA的提取使用的RNeasy微型试剂盒（Qiagen）的cDNA使用qScript cDND合成试剂盒（广达生物科技公司生产）

<ol>
	<li>Wielowiejski, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ueber+das+blutgewebe+der+insekten.">Ueber das blutgewebe der insekten.</a> Zeit. Wiss. Zool. 43, 512-536 .</li><li>Krupp, J. J., Kent, C., Billeter, J. C., Azanchi, R., So, A. K., Schonfeld, J. A., Smith, B. P., Lucas, C., Levine, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Social+experience+modifies+pheromone+expression+and+mating+behavior+in+male+Drosophila+melanogaster.">Social experience modifies pheromone expression and mating behavior in male Drosophila melanogaster.</a> Curr. Biol. 18, 1373-1383 (2008).</li><li>Locke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+membranes,+Golgi+complexes,+and+vacuolar+systems.">Surface membranes, Golgi complexes, and vacuolar systems.</a> Annu. Rev. Entomol. 48, 1-27 (2003).</li><li>Billeter, J. C., Atallah, J., Krupp, J. J., Millar, J. G., Levine, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specialized+cells+tag+sexual+and+species+identity+in+Drosophila+melanogaster.">Specialized cells tag sexual and species identity in Drosophila melanogaster.</a> Nature. 461, 987-991 (2009).</li><li>Gould, A. P., Elstob, P. R., Brodu, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+oenocytes:+a+model+system+for+studying+cell-fate+specification+by+Hox+genes.">Insect oenocytes: a model system for studying cell-fate specification by Hox genes.</a> J. Anat. 199, 25-33 (2001).</li><li>Lawrence, P. A., Johnston, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+of+the+Drosophila+abdomen:+the+epidermis,+oenocytes+and+ventral+muscles.">Cell lineage of the Drosophila abdomen: the epidermis, oenocytes and ventral muscles.</a> J. Embryol. Exp. Morphol. 72, 197-208 (1982).</li><li>Johnson, M. B., Butterworth, 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=Maturation+and+aging+of+adult+fat+body+and+oenocytes+in+Drosophila+as+revealed+by+light+microscopic+morphometry.">Maturation and aging of adult fat body and oenocytes in Drosophila as revealed by light microscopic morphometry.</a> J. Morphol. 184, 51-59 (1985).</li><li>Gutierrez, E., Wiggins, D., Fielding, B., Gould, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specialized+hepatocyte-like+cells+regulate+Drosophila+lipid+metabolism.">Specialized hepatocyte-like cells regulate Drosophila lipid metabolism.</a> Nature. 445, 275-280 (2007).</li><li>Marcillac, F., Bousquet, F., Alabouvette, J., Savarit, F., Ferveur, 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=A+mutation+with+major+effects+on+Drosophila+melanogaster+sex+pheromones.">A mutation with major effects on Drosophila melanogaster sex pheromones.</a> Genetics. 171, 1617-1628 (2005).</li><li>Chertemps, T., Duportets, L., Labeur, C., Ueyama, M., Wicker-Thomas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+female-specific+desaturase+gene+responsible+for+diene+hydrocarbon+biosynthesis+and+courtship+behaviour+in+Drosophila+melanogaster.">A female-specific desaturase gene responsible for diene hydrocarbon biosynthesis and courtship behaviour in Drosophila melanogaster.</a> Insect Mol. Biol. 15, 465-473 (2006).</li><li>Legendre, A., Miao, X. X., Da Lage, J. L., Wicker-Thomas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+a+desaturase+involved+in+female+pheromonal+cuticular+hydrocarbon+biosynthesis+and+courtship+behavior+in+Drosophila.">Evolution of a desaturase involved in female pheromonal cuticular hydrocarbon biosynthesis and courtship behavior in Drosophila.</a> Insect. Biochem. Mol. Biol. 38, 244-255 (2008).</li><li>Shirangi, T. R., Dufour, H. D., Williams, T. M., Carroll, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+evolution+of+sex+pheromone-producing+enzyme+expression+in+Drosophila.">Rapid evolution of sex pheromone-producing enzyme expression in Drosophila.</a> PLoS Biol. 7, e1000168-e1000168 (2009).</li></ol>

在这个视频文章中，我们提出了详细的解剖协议，准备从成人oenocytes D. 果蝇的方式适用于分子生物学分析。其他地方2，解剖方法的一个缩写文本为基础的帐户已被描述和oenocyte准备已被证明是适合提取的RNA和蛋白 2 。使用该制剂，它可能也有可能发展explanted oenocytes培养方法。 oenocyte植体文化协议将有助于调查目的是，确定这些细胞的代谢能力的自主的合成和分泌的?...

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<td>Sylgard</td>
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dissection of oenocytes from adult drosophila melanogaster

在<em&gt;果蝇</em&gt;，在其他昆虫一样，上外表面的角质层的蜡质层，主要由烃类化合物，对干燥和其他环境挑战的保护。几个这些表皮烃（CHC）的化合物也可作为semiochemical信号，并作为调解同一物种的成员之间的信息素通讯，或在某些情况下，不同物种之间，并影响人们的行为。简称为oenocytes专门的细胞被视为CHC合成的主站点。但是，相对鲜为人知的是关于oenocytes参与的生物合成，运输，和沉积的途径，促进CHC输出的调控。鉴于社区卫生中心在昆虫生物学的几个方面，包括化学通讯，干燥性和免疫力，发挥的重​​要作用，重要的是要获得一个更好地理解这些专门的细胞内的分子和基因调控CHC生产。成人oenocytes<em&gt; D。果蝇</em位于腹部包膜，metamerically摆着彩带般沿内各腹节的表皮表面辐射的集群。在这个视频文章中，我们展示了一个夹层技术用于准备从成人的oenocytes<em&gt; D。果蝇</em&gt;。具体来说，我们提供了详细的一步一步的示范（1）如何圆角准备成人<em&gt;果蝇</em&gt;腹部，（2）如何识别oenocytes和辨别其他组织，和（3）如何从腹部包膜完好oenocyte集群。包含一个简短的如何该制剂可用于检查碳氢合成有关的基因表达的实验说明。解剖的准备表明此处将允许在成年果蝇的oenocyte功能的详细的分子和遗传分析。

Watch this Scientific Journal Video about Dissection of Oenocytes from Adult Drosophila melanogaster at JoVE.com

Dissection of Oenocytes from Adult Drosophila melanogaster

在昆虫中，oenocytes产生表皮碳氢化合物。这些化合物防止干燥和促进化学通讯。在这里，我们展示了一个夹层的技术，用来隔离从成人oenocytes<em&gt;果蝇</em&gt;，并说明如何可以利用该制剂的研究碳氢合成有关的基因。

从成人Oenocytes剖析果蝇</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

dissection-of-oenocytes-from-adult-drosophila-melanogaster

Research

JoVE Journal

Biology

Dissection of Oenocytes from Adult Drosophila melanogaster

20.4K 次观看.  University of Toronto. 在昆虫中，oenocytes产生表皮碳氢化合物。这些化合物防止干燥和促进化学通讯。在这里，我们展示了一个夹层的技术，用来隔离从成人oenocytes果蝇，并说明如何可以利用该制剂的研究碳氢合成有关的基因。

视频: Dissection of Oenocytes from Adult Drosophila melanogaster

Dissection of Drosophila Ovaries

剖析果蝇卵巢

科研

生物学

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

在果蝇幼虫中枢神经系统的剖析

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

从3龄果蝇幼虫的成虫光盘夹层

Whole Cell Recordings from Brain of Adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown.  The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.

This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording.

从整个成年果蝇大脑细胞记录

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

高分辨率视频跟踪移动的，在成年果蝇。

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

从成年小鼠海马齿状回的剖析

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

剖析器官的成年斑马鱼

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

分离果蝇睾丸

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

注射胰岛素和血淋巴的提取来衡量成人胰岛素敏感性果蝇</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

解剖和免疫染色成虫盘的<em&gt;果蝇</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...