Name: labeling of single cells in the central nervous system of drosophila melanogaster
Uploaded: 2013-03-04T12:00:00
Description: Watch this Scientific Journal Video about Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster at JoVE.com

这项工作是支持的赠款从DFG GMT

在我们的实验室，我们用了两个略有不同的变体的单神经元标记法。差异有关胚胎收集，dechorionisation，devitellinisation和胚胎圆角步骤。 图1给出了一个变体的共同和不同的步骤概述。 1。微针的制备染料注射胚胎解剖和微量<ol><li>胚胎解剖的玻璃针都来自玻璃毛细管（直径1 mm和0.1 mm壁厚）与萨特拉出器（科学仪器）或类似的设备。或者，一个电解削尖0.15毫米的?...

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果蝇作为一个模型系统的一个主要优点是，它允许单细胞水平上的发展和功能的分析。有关的神经系统，其中的细胞类型的多样性是非常高的，周边小区的功能和形态，可以是完全不同的，这是特别有用的。 我们在这里提出的方法允许单个神经元的标记的染料，可以被转化为一个永久染色或直接用荧光显微镜检查。它揭示了神经过程非常详细的形态（ 图4）。</s...

在单个细胞水平的神经元形态的知识是理解神经元的连接和中枢神经系统功能的一个重要先决条件。因此，从最早的天，神经科学，研究人员试图开发单细胞标记技术（参见图7为处理这一问题的历史）。经典方法，如高尔基染色的神经元的形态，但提供了极好的分辨率，是不适合的，如果一个人来标记特定类型的神经元的定向的方式，作为染色以随机方式发生。染色单细胞细胞内或juxtacellular的注射染料从一个微电极的方法的发展，处理特定的标记的要求。 单个神经元的染料注射到果蝇的应用提出了重大挑战，因为规模小的有机体，它的神经元。然而，单一的胚胎果蝇的神经元染色</em&gt;神经元实现了在20世纪80年代中期，在实验室中科古德曼15。虽然长足的方法有用的，在过去的20-30年（ 例如 ，10）在果蝇神经元的发育机制，提供了一些重要的见解，很多工人纷纷避开它，主要是因为它的技术要求。 基因技术在果蝇的神经元标签的情况在最近几年也促进了不受欢迎的单个神经元的染料注入。 GAL4表达的膜有针对性的GFP结构，可提供出色的分辨率神经形态1，20。然而，这种方法有一定的局限性：经常不可避免的多个单元格中的GFP表达，可以掩盖单个神经元的结构和一个GAL4驱动线可能不是可用来驱动在感兴趣的特定的神经元中的表达。 MARCM（马赛克分析与代理商ressible细胞标记物）方法8基本上任何神经元标记可以提供在单个细胞水平，但不能被成功地用于在胚胎和早期幼虫，因为的GAL80蛋白的周转缓慢。 由于这些遗传标记的局限性，我们相信在果蝇胚胎，单个神经元的染料注入仍然是一个有价值的技术，值得更广泛的应用。为了促进这一目标，我们在这里提供的方法的详细说明。说明它的权力是由我们最近帐户的形态一套完整的interneurons在腹部neuromeres的果蝇胚胎后期14。本研究揭示的形态变异的个体的神经细胞类型和组织在中枢神经系统中的胚胎neuromere的原则，将不可能有任何其他现有的标记方法。

<table border="1"><tbody><tr><td> 的试剂/材料名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td></td><td></td><td></td><td> 试剂 </td></tr><tr><td> DAB </td><td> Sigma-Aldrich公司</td><td> <a href="http://www.sigmaaldrich.com/catalog/product/sigma/d5905" target="_blank">D-5905</a> </td><td>在100mM的Tris-HCl，pH值7.4的2-3毫克/毫升</td></tr><tr><td> DiI荧光</td><td> Invitrogen公司/分子探针</td><td> <a href="http://products.invitrogen.com/ivgn/product/D282" target="_blank">D-282</a> </td><td>在乙醇中的1毫克/毫升</td></tr><tr><td> <a href="http://www.merckmillipore.com/germany/chemicals/formaldehydloesung-min-37-prozent/MDA_CHEM-104002/p_Wt6b.s1L6pAAAAEWpOEfVhTl" target="_blank">甲醛</a> </td><td>默克密理博</td><td></td><td>在PBS 7.4％ </td> &lt;/ TR&gt; <tr><td>甘油</td><td> <a href="http://www.carlroth.com/catalogue/" target="_blank">罗斯</a> </td><td> 3783 </td><td>在PBS中的70％的</td></tr><tr><td>正庚烷胶</td><td>拜尔斯道夫公司</td><td>大提琴31-39-30 * </td><td>稀释约。 1为1，n-庚烷</td></tr><tr><td> PBS </td><td></td><td></td><td> 1个</td></tr><tr><td> TRIS </td><td> <a href="http://www.carlroth.com/catalogue/" target="_blank">罗斯</a> </td><td> 4855 </td><td></td></tr><tr><td> Vectashield </td><td>矢量实验室</td><td> <a href="http://www.vectorlabs.com/catalog.aspx?prodID=427" target="_blank">H1000</a> </td><td></td></tr><tr><td></td><td></td><td></td><td> 设备 </td></tr><tr><td>共聚焦显微镜</td><td>徕卡</td><td> TCS SP2 </td><td>视图和文档标号的荧光</td></tr><tr><td> DC -mplifier </td><td>德拉甘公司</td><td>基础ION-100 </td><td>执行标号</td></tr><tr><td>盖玻片</td><td> <a href="http://www.menzel.de/" target="_blank">门泽尔</a> </td><td> BB018018A1 </td><td> 18×18毫米</td></tr><tr><td>盖玻片</td><td> <a href="http://www.menzel.de/" target="_blank">门泽尔</a> </td><td> BB024060A1 </td><td> 24×60毫米</td></tr><tr><td>解剖显微镜</td><td>徕卡</td><td> MZ8 </td><td>准备，剖析胚胎的</td></tr><tr><td>平板毛细管</td><td> <a href="http://www.hilgenberg-gmbh.de/" target="_blank">Hilgenberg</a> </td><td></td><td>外直径1毫米; GLAS厚度为0.1毫米</td></tr><tr><td>注射毛细管</td><td>科学产品</td><td> GB 100 TF 8P </td><td></td></tr><tr><td>微操作机器人ŕ </td><td>徕卡</td><td></td><td>执行标号</td></tr><tr><td>型号：P-97 </td><td>萨特仪器</td><td></td><td>拉毛细血管</td></tr><tr><td>对象的幻灯片</td><td> <a href="http://www.marienfeld-superior.com/" target="_blank">马林费尔德高级</a> </td><td> 1000000 </td><td></td></tr><tr><td>科学显微镜</td><td>蔡司</td><td> Axioskop 2电机</td><td>查看标号后光转化</td></tr><tr><td>科学显微镜</td><td>奥林巴斯</td><td> BX50 </td><td>执行标号</td></tr><tr><td>索尼MC3255摄像机</td><td>索尼/ AVT喇叭</td><td>索尼MC3255 </td><td>光电转换后记录标号</td></tr></tbody></table> 表1中。

labeling of single cells in the central nervous system of drosophila melanogaster

在这篇文章中，我们将介绍如何单独标记的亲脂性荧光膜标记DII juxtacellular注射在果蝇胚胎中枢神经系统的神经元。这种方法可以非常详细的神经细胞形态的可视化。这是可能的标记在中枢神经系统中的任何单元格：在DIC的光学系统或通过表达的荧光（如GFP）的遗传标记的目标神经元的细胞体的可视化。 DiI荧光标记后，可以被转化为一个永久褐变光转化用透射光与DIC光学允许可视化的细胞形态。或者，可以直接观察到DiI荧光标记的细胞用激光共聚焦显微镜，使要colocalised基因引入荧光报告蛋白。这项技术可以被用在任何动物，不论基因型，使得有可能在单细胞分辨率分析突变体的表型。

图4示出了该技术的典型的结果，在这里，我们将描述图4A示出一个DiI荧光填充的单interneuron，干净光转换的一个例子。它很好地体现了这些准备工作提供大量的细节。下观看时，DIC的光学标记的单元格内的非标记的周围组织的空间范围内变为可见的， 例如 ，所述单元主体的内皮层和内神经纤维的纤维突起的位置。 图4B是染料下降...

Watch this Scientific Journal Video about Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster at JoVE.com

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster (Video) | JoVE

我们提出了一种技术，用于标识单个神经元在中枢神经系统（CNS）的<em&gt;果蝇</em&gt;，胚胎，它允许由任一透射光或激光共聚焦显微镜的神经元的形态学分析。

标记的单细胞在中枢神经系统<em&gt;果蝇</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

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Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

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