IM是由居里夫人重新整合的赠款支持。这项工作是由以色列科学基金资助没有的支持。 1146年至1108年，由海伦和马丁金梅尔在魏茨曼科学研究所的干细胞研究和Leir慈善基金会。

1。产卵<ol><li> 5天前清扫：允许交配女性躺在鸡蛋2-4个小时，用酵母补充的新鲜食品。为了得到同步和发达的幼虫，重要的是不要有人满为患培养（约30卵/25毫米瓶）。通常情况下，每小瓶7-16女性，取决于他们躺在。 </li></ol> 2。选择幼虫<ol><li>准备9以及玻璃，夹层菜充满林格氏液（128毫米氯化钠，2MM KCL，1.8MM，4MM氯化钙氯化镁 2，35.5mM蔗糖，5MM HEPES pH值6.9） 。 </li><li>准备在6孔林格的中期和它放在冰盘细胞过滤器。另外，我们用一个尼龙网装上特制的模具。 </li><li>选择使用罚款（钳）生物镊子从小瓶墙壁定时幼虫和林格含解剖菜。 </li><li>一名女幼虫转移到一个干净的井。我们区别于其性腺男性的女性。男性睾丸很容易识别嵌入在脂肪体后的第三大明确椭圆。女性卵巢，位于肥胖的身体的同一部分，可以被认定为一个更小的，明确的，全面领域。 </li></ol> 3。剖析幼虫<ol><li>保持幼虫仅下跌后大脑与钳和删除的第二双钳头。 </li><li>广场上的背侧后剩余的部分，气管朝下。 </li><li>握住一双镊子后气孔的幼虫，然后向内推缓慢而另一对角质层向后滑动，直到大约一半的幼虫的脂肪体出现。 </li><li>牢牢把握的后部，使用产钳的另一对松散举行的角质层。轻轻地，慢慢地绝尘而去后，使差距的角质层，并通过附加肠道幻灯片。在这个过程结束时，应完全分离，脂肪体，从肠道内和角质层。肠道脂肪体前部断开。为了使染色和安装容易，重要的是，肥胖的身体将保持不变。 </li><li>湿与林格中型牧场吸管彻底，最好是从一个含有解剖幼虫。这有助于大衣移液器和防止肥胖的身体，从坚持它。使用牧场吸管转移到肥胖的身体在冰冷的林格氏细胞过滤器中的介质。 </li></ol> 4。固定和染色所有步骤都是在室温下进行，除了第一抗体的潜伏期，在4 ° C。 <ol><li>孵育5％甲醛林格的中期脂肪体。轻轻摇动20分钟。 </li><li>用1％的PBT（1％的Triton X - 100的PBS）洗5分钟。重复此步骤10分钟和45分钟的第三次。 1％的Triton X - 100的穿孔幼虫的卵巢和允许抗体渗透。 </li><li> PBTB 0.3％（0.3％的Triton X - 100和1％的牛血清白蛋白的PBS）为1小时，轻轻摇动座</li><li>所需的第一抗体在0.3％，超过晚上PBTB稀释后在4 °与轻轻摇动孵育。这一步通常在0.2毫升管上滚。 </li><li>转移脂肪体细胞过滤器。 </li><li>洗3次，每次30分钟，轻轻摇动PBTB 0.3％。 </li><li>与0.3％，5％的正常驴血清轻轻摇动1小时补充PBTB座。 </li><li>孵育一个适当的二级抗体稀释于封闭液（0.3％与5％，0.3％，PBT的正常驴血清补充PBTB）（根据制造商的规范）为2小时，轻轻摇动。如果抗体荧光，在黑暗从这个点开始孵化。这一步通常在0.2毫升管上滚。 </li><li>洗3次，每次30分钟，用0.​​3％轻轻摇动PBT。 </li></ol> 5。安装<ol><li>脂肪体转移到一个干净的Eppendorf管中。非常小心地取出所有的液体，立即盖上Vectashield安装媒体。我们经常使用vectashield因为它不会变硬，而卵巢被脂肪体分离。 样品可以保存在4 ° C长达一个星期的安装媒体。 </li><li>剪下一个枪头，并用它来仔细安装媒体的最小量（约30μL为30毫米的覆盖防滑）转移到载玻片的脂肪体。 </li><li>使用镀镍针持有人，持有直径0.1毫米引脚摊开肥胖的身体。卵巢是位于后三分之一的肥胖的身体。他们周围的脂肪体，通常是形如一个“花”。这有助于确定卵巢的位置。解剖出肥胖的身体的其余部分的“花朵”，并放弃后者。 </li><li>去除脂肪的身体surrounding仔细越过它周围的两个引脚性腺。将在幻灯片上，远离脂肪体残留的孤立性腺。 </li><li>盖上盖玻片，指甲油密封。 </li><li>可视化直接使用共聚焦显微镜。样品可以保存在4 ° C至三个星期。 </li></ol> 6。代表性的成果我们已经使用了上述协议，按照建立体卵巢内的细胞谱系，包括GSCs和他们的体龛。为此，我们使用的特异性抗体和标记以区分不同类型的细胞在发展中国家性腺。在这里，我们显示的两个LL3染卵巢抗体的不同组合例子。图1A突出终端长丝和帽细胞（绿色），它们共同形成的利基体细胞。图1B，显示混合细胞（ICS，洋红），直接接触生殖细胞（蓝色）。 <img src="/files/ftp_upload/2537/2537fig1.jpg" alt="图1" /> 图1。LL3卵巢。 （一）单克隆抗体1B1（洋红色），概述了体细胞和污渍fusome，内PGCS细胞内的细胞器（箭头）。陷阱HH - lacZ的增强（抗β-半乳糖苷酶，绿色）表示在终端的细丝。在基地的丝体第细胞（箭头）可以观察到。 （二）1B1抗体（绿色）列出所有的体细胞在卵巢。反瓦萨（蓝色）标签所有PGCS。 PGCS直接接触（集成电路，反堵车，洋红）的混合细胞。酒吧（A和B）是20微米。

<ol>
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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+niche+maintaining+germ+line+stem+cells+in+the+Drosophila+ovary.">A niche maintaining germ line stem cells in the Drosophila ovary.</a> Science. , 290-328 (2000).</li><li>Guo, Z., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glypican+Dally+is+required+in+the+niche+for+the+maintenance+of+germline+stem+cells+and+short-range+BMP+signaling+in+the+Drosophila+ovary.">The glypican Dally is required in the niche for the maintenance of germline stem cells and short-range BMP signaling in the Drosophila ovary.</a> Development. 136, 3627-3635 (2009).</li><li>Wang, X., Harris, R. E., Bayston, L. J., Ashe, H. 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本视频演示晚三龄幼虫卵巢的隔离和染色协议。要经常和可靠地执行这一协议，应注意以下几点： <ol><li>同步和发达的幼虫，较拥挤，必须避免。 </li><li>为了避免损失小的半透明卵巢，确保解剖脂肪体完好无损。这也将有助于在安装阶段的定位卵巢，尤其是当安装年​​轻（第二或第三龄开始）卵巢。 </li><li>为了同时处理很多样品，使用细胞过滤器或其他的玩意儿。 </li><li>确保永不干涸的样...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>J.T.Baker</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Kcl</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2</td>
<td>&nbsp;</td>
<td>Merck</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>J.T.Baker</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Hepes</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Albumin Bovine Fraction V</td>
<td>&nbsp;</td>
<td>MP Biomedicals</td>
<td>160069</td>
<td>&nbsp;</td>
<tr>
<td>Dumont biology tweezers 5 dumstar polished</td>
<td>&nbsp;</td>
<td>FST Fine Science Tools</td>
<td>11295-10</td>
<td>&nbsp;</td>
<tr>
<td>Nickel plated pin holder</td>
<td>&nbsp;</td>
<td>FST Fine Science Tools</td>
<td>26018-17</td>
<td>&nbsp;</td>
<tr>
<td>s.s minutien pins 0.1mm diam, 10mm long</td>
<td>&nbsp;</td>
<td>FST Fine Science Tools</td>
<td>26002-10</td>
<td>&nbsp;</td>
<tr>
<td>9 well plates 85X100 mm, 22mm o.d.x7mm deep</td>
<td>&nbsp;</td>
<td>CORNING</td>
<td>7220-85</td>
<td>&nbsp;</td>
<tr>
<td>Stereo Microscope MZ 16.5 with a standard transmitted light base TL ST</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>6 well plates</td>
<td>&nbsp;</td>
<td>Costar</td>
<td>3516</td>
<td>&nbsp;</td>
<tr>
<td>Slides</td>
<td>&nbsp;</td>
<td>Menzel Glaser GmbH</td>
<td>798</td>
<td>&nbsp;</td>
<tr>
<td>Cover slips</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>2940-223</td>
<td>&nbsp;</td>
<tr>
<td>Mounting media</td>
<td>&nbsp;</td>
<td>Vectashield</td>
<td>H-1200</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>FAL352350</td>
<td>&nbsp;</td>
<tr>
<td>1B1 antibody</td>
<td>&nbsp;</td>
<td>Developmental Studies Hybridoma Bank</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Traffic Jam</td>
<td>&nbsp;</td>
<td>Laboratory of Dr. Dorothea Godt</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Anti-Vasa</td>
<td>&nbsp;</td>
<td>Laboratory of Dr. Ruth Lehmann</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Anti &beta;-Galactosidase</td>
<td>&nbsp;</td>
<td>Cappel</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Secondary Antibodies</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

dissection and staining of drosophila larval ovaries

许多器官取决于其发展的干细胞，胚胎发育过程中和在成年生活中的保养或维修。因此，了解如何干细胞的形式，以及如何与环境互动的了解发展，动态平衡和疾病的关键。曾担任果蝇卵巢生殖系干细胞与他们的躯体支持细胞（利基）1，2（GSCs）的相互作用的一个有影响力的模型。的利基和GSCs已知的位置，再加上基因操纵他们的能力，使研究人员阐明了各种干细胞及其龛3-12之间的相互作用。 尽管控制GSC维护和分化机制的信息财富，相对鲜为人知的是，如何GSCs和他们的体龛在发展过程中形成。约18体龛，其细胞成分，包括终端长丝和帽细胞（图1），在第三幼虫龄 13-17形式。 GSCs源于原始生殖细胞（PGCs）。 PGCS增殖早期幼虫阶段，但形成的利基的PGCS分组成为GSCs 7，16，18，19。总之，体利基细胞的GSCs一个功能单位，生产鸡蛋整个生物体的寿命。 就形成的GSC单位许多问题仍然悬而未决。如壁龛和干细胞前体的前体细胞，或代内PGCS不对称，因为他们成为GSCs之间的协调过程，最好是能在幼虫研究。然而，有条不紊幼虫卵巢发育的研究是身体具有挑战性。首先，幼虫卵巢小。即使在后期幼体阶段，他们只有100μm的跨越。此外，卵巢是透明的，嵌入在白色脂肪组织。在这里，我们描述一个孤立晚三龄（LL3） 果蝇幼虫，用荧光抗体染色，卵巢的一步一步的协议。我们提供了一些问题，如定位卵巢，染色和洗涤不下沉的组织，确保抗体渗透到组织的技术解决方案。该协议可应用于早期幼虫期和幼虫睾丸以及。

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

Dissection and Staining of Drosophila Larval Ovaries

壁龛和干细胞如何在发展过程中形成的，是一个具有实际意义的重要问题。在<em&gt;果蝇</em&gt;卵巢，生殖系干细胞和体龛的形式在幼虫发育。本视频演示了如何解剖，染色和安装从晚三龄（LL3）女性性腺<em&gt;果蝇</em&gt;幼虫。

夹层和染色果蝇幼虫卵巢

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

dissection-and-staining-of-drosophila-larval-ovaries

Research

JoVE Journal

Biology

Dissection and Staining of Drosophila Larval Ovaries

26.1K 次观看.  Weizmann Institute of Science. 壁龛和干细胞如何在发展过程中形成的，是一个具有实际意义的重要问题。在果蝇卵巢，生殖系干细胞和体龛的形式在幼虫发育。本视频演示了如何解剖，染色和安装从晚三龄（LL3）女性性腺果蝇幼虫。

视频: Dissection and Staining of Drosophila Larval Ovaries

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 ...

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

果蝇幼虫NMJ夹层

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

果蝇幼虫NMJ免疫

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

现场剖析果蝇胚胎抗体染色筛选突变体集合的简化方法

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

幼虫，少年和成年斑马鱼的心脏解剖，斑马鱼</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and organogenesis. Immunostaining allows for the visualization of developing tissues and organs. However, a protective cuticle that forms at the end of embryogenesis prevents permeation of antibodies into late-stage embryos and larvae. While dissection prior to immunostaining is regularly used to analyze Drosophila larval tissues, it proves inefficient for some analyses because small tissues may be difficult to locate and isolate. Sonication provides an alternative to dissection in larval Drosophila immunostaining protocols. It allows for quick, simultaneous processing of large numbers of late-stage embryos and larvae and maintains in situ morphology. After fixation in formaldehyde, a sample is sonicated. Sample is then subjected to immunostaining with antigen-specific primary antibodies and fluorescently labeled secondary antibodies to visualize target cell types and specific proteins via fluorescence microscopy. During the process of sonication, proper placement of a sonicating probe above the sample, as well as the duration and intensity of sonication, is critical. Additonal minor modifications to standard immunostaining protocols may be required for high quality stains. For antibodies with low signal to noise ratio, longer incubation times are typically necessary. As a proof of concept for this sonication-facilitated protocol, we show immunostains of three tissue types (testes, ovaries, and neural tissues) at a range of developmental stages.

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.

后期阶段的胚胎和幼虫的超声，促进免疫荧光染色<em&gt;果蝇</em&gt;组织<em&gt;原位</em

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and ...

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 ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

解剖和安装的<em&gt;果蝇</em&gt;蛹眼光盘

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

幼虫活动的测量<em&gt;果蝇</em&gt;活动监视器

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...