Name: application of multicolor flpout technique to study high resolution single cell morphologies and cell interactions of glia in drosophila
Uploaded: 2017-10-20T14:00:00
Description: Watch this Scientific Journal Video about Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila at JoVE.com

作者感谢 Arnim Jenett, Aljoscha 海滨, 和鲁宾实验室的其他成员的建议和共享的未发表的试剂和 Janelia 飞光项目组产生共焦图像。作者还感谢高卢实验室的成员对手稿的评论。

1. 为多色 FlpOut (MCFO) 实验准备苍蝇 注意: MCFO 技术指的是所谓的 Flp 介导的停止盒式切除 (FlpOut) 的修改版本。转基因 MCFO 蝇携带热休克启动子 (hsp)-Flp 重组和不同的记者在无人控制下。每个记者都由一个 myristoylated (myr) 超级文件夹绿色荧光蛋白 (sfGFP) 的共同骨干组成, 其中10个表位标签 ( e. g ., HA, 旗或 V5) 的副本已经插入。所产生的荧光蛋白的命名和 #34; 意大利面条怪兽?...

<ol>
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该协议描述了一个简单和有效的方法来研究在高分辨率的兴趣组织内的不同细胞类型的形态学。采用 MCFO 技术, 多种不同表位标记的记者组合使用, 用于多色随机标记 (图 2)。与其他方法类似, 如 Brainbow/Flybow15,16,17, MCFO 通过标记共增加了标签的多样性, 从而使细胞边界的可视化成为细胞间相互作用的研究。?...

神经胶质是神经系统的神经细胞细胞, 长期以来被认为为神经元提供了一个静止的框架, 因此没有得到详细的研究。然而, 在人类中, 胶质细胞在 NS (约 90%) 中占绝大多数, 并分成几个不同的类别, 包括星状细胞、突、小胶质细胞和雪旺电池。在果蝇中, 胶质细胞在 NS 中占10% 左右。有趣的是, 它们的形态和功能与脊椎动物的1,2中发现的非常相似。它们的形态包括血脑屏障 (BBB) 形成上皮细胞、鞘层和星形星状胞。
果蝇中枢神经系统 (CNS) 包括以下主要结构: 包含神经元细胞体的皮层区域;neuropils 那海港突触连接;连接不同 neuropiles 的小的和大的轴突束;与中枢神经系统连接感觉器官和肌肉的外周神经 (图 1)。胶质细胞被发现与所有这些解剖结构相关: 皮质胶质细胞 (CG) 在皮层区, 星形胶质细胞和鞘胶质细胞 (如) 在 neuropile 区, 鞘胶质细胞也与中央轴突和周边神经 (地名), 最后, 两个片状胶质细胞, 膜胶质细胞 (PG) 和 subperineurial (), 其中一起形成一个连续的层覆盖整个 NS (图 2)。
以往的研究表明, 胶质细胞在 NS 的发展中起着重要的作用。它们通过对系统循环的胰岛素样肽进行反应来监测神经细胞的数量, 为神经元提供营养支持, 如星形胶质细胞-神经元乳酸梭, 并通过吞噬3,4来消除死亡神经元,5,6. 在成熟的 ns 中, 胶质细胞维持血脑屏障, 接受神经递质并维持离子稳态, 作为主要的免疫单元在 ns, 因为巨噬细胞不能破坏 bbb, 并调节突触活动以及动物行为6,7,8,9,10,11。
不同的胶质亚型是否具有特殊功能仍然是一个重要的开放性问题。然而, 对胶质细胞进行系统的全基因组分析, 特别是在成人身上, 由于缺乏适当的基因工具来进行操作而受到阻碍。在这里, 一个方法, 使细胞形状的有效和容易的表征研究复杂的细胞间的相互作用提出。这项技术已被用于表征不同的胶质亚型在成人果蝇脑中的形态学, 但是, 根据具体的 GAL4 驱动程序, 它可以适应研究神经元12,13, 任何类型的混合细胞, 原则上任何发展阶段的任何组织。

<table>
	<tbody>
		<tr>
			<td>Water bath</td>
			<td>Grant</td>
			<td>GD100</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>Sarstedt</td>
			<td>72.737.002</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting dish 30 mm x 12 mmm</td>
			<td>Electron Microscopy Sciences</td>
			<td>70543-30</td>
			<td>Glass dissection dish</td>
		</tr>
		<tr>
			<td>Pyrex 3 Depression Glass Spot Plate</td>
			<td>Corning</td>
			<td>7223-34</td>
			<td>Glass dissection plates</td>
		</tr>
		<tr>
			<td>Sylgard Black</td>
			<td>SYLGARD, Sigma-Aldrich</td>
			<td>805998</td>
			<td>home made with charcoal</td>
		</tr>
		<tr>
			<td>ExpressFive S2 cell culture medium</td>
			<td>Invitrogen</td>
			<td>10486-025</td>
			<td></td>
		</tr>
		<tr>
			<td>20% PFA</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Roth</td>
			<td>3051.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Jackson Laboratories</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Jackson Laboratories</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit HA-tag</td>
			<td>Cell Signaling</td>
			<td>C29F4</td>
			<td>Primary antibody, dilution 1:500</td>
		</tr>
		<tr>
			<td>Rat FLAG-tag</td>
			<td>Novus Biologicals</td>
			<td>NBP1-06712</td>
			<td>Primary antibody, dilution 1:100</td>
		</tr>
		<tr>
			<td>Mouse V5-tag:DyLight 549</td>
			<td>AdSerotec</td>
			<td>0411</td>
			<td>Conjugated antibody, dilution 1:200</td>
		</tr>
		<tr>
			<td>anti-rabbit AlexaFluor 488</td>
			<td>Invitrogen</td>
			<td>A11034</td>
			<td>Secondary antibody, dilution 1:250</td>
		</tr>
		<tr>
			<td>anti-rat DyLight 647</td>
			<td>Jackson Laboratories</td>
			<td>712-605-153</td>
			<td>Secondary antibody, dilution 1:100</td>
		</tr>
		<tr>
			<td>Vecta Shield</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>SlowFate Gold</td>
			<td>Invitrogen</td>
			<td>S36937</td>
			<td></td>
		</tr>
		<tr>
			<td>Secure Seal Spacer</td>
			<td>Grace Biolabs</td>
			<td></td>
			<td>Contact company for ordering</td>
		</tr>
		<tr>
			<td>Microscope cover glass 22 X 60 mm</td>
			<td>Marienfeld</td>
			<td>101152</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass 22 x 22 mm</td>
			<td>Roth</td>
			<td>H874</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope, Leica MZ6</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope LSM710</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Immersol</td>
			<td>Zeiss</td>
			<td>518 F</td>
			<td>Immersion oil for fluorescence-microscopy, halogen free</td>
		</tr>
		<tr>
			<td>Immersol</td>
			<td>Zeiss</td>
			<td>W 2010</td>
			<td>Immersion fluid for water-immersion objectives, halogen free</td>
		</tr>
		<tr>
			<td>R56F03-GAL4 (EG)</td>
			<td>Bloomington Stock Center</td>
			<td>39157</td>
			<td>GAL4 driver</td>
		</tr>
		<tr>
			<td>R86E01-GAL4 (ALG)</td>
			<td>Bloomington Stock Center</td>
			<td>45914</td>
			<td>GAL4 driver</td>
		</tr>
		<tr>
			<td>hspFlpPestOpt; UAS-FRT-stop-FRT-myr-smGFP-HA, UAS-FRT-stop-FRT-myr-smGFP-FLAG, UAS-FRT-stop-FRT-myr-smGFP-V5</td>
			<td>Bloomington Stock Center</td>
			<td>64085</td>
			<td>UAS reporter (https://bdscweb.webtest.iu.edu/stock/misc/mcfo.php)</td>
		</tr>
		<tr>
			<td>Fiji (Image J)</td>
			<td></td>
			<td></td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>Multi Time Macro</td>
			<td>Zeiss</td>
			<td></td>
			<td>Software for automated scanning</td>
		</tr>
	</tbody>
</table>

application of multicolor flpout technique to study high resolution single cell morphologies and cell interactions of glia in drosophila

细胞表现出不同的形态和复杂的解剖关系。细胞如何与邻居互动？单元格类型之间的交互是否不同, 甚至在给定类型中也有所不同？他们遵循什么样的空间规则？到目前为止, 由于缺乏用于高分辨率单细胞标记的工具, 这些基本问题的答案在体内受到了阻碍。在这里, 提供了一个详细的协议目标单细胞与多色 FlpOut (MCFO) 技术。这种方法依赖于三不同标记的记者 (HA, 旗帜和 V5) 在无人控制, 保持沉默的转录终结者两侧的两个首次登记税网站 (首次登记-停止-首次登记税)。热休克脉冲诱发热休克诱导的 Flp 重组的表达, 它在单个单元格中随机删除了首次登记停用的卡带: 表达仅发生在同时表达 GAL4 驱动程序的单元格中。这将导致一个给定单元格类型的不同颜色的单元格数组, 它允许在高分辨率下可视化单个细胞形态。作为一个例子, MCFO 技术可以结合特定的胶质 GAL4 驱动, 以可视化的形态, 不同的胶质亚型在成年果蝇脑。

本节说明了在成人果蝇脑中使用 MCFO 技术可以获得的结果的示例。 图 3显示了该方法的示意图。三不同的膜标记的记者 (myr-smGFP-HA, myr-smGFP-旗和 myr-smGFP-V5) 在无人控制系统下, 由两个首次登记税网站 (首次登记-停止-首次登记税) 两侧的转录终结者保持沉默。热休克脉冲诱导 Flp 重组的表达, 它在单个单元格中随机删除了首次登记停用...

Watch this Scientific Journal Video about Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila at JoVE.com

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

细胞表现出不同的形态, 并与它们的邻居建立了各种相互作用。该协议描述了如何揭示单细胞的形态学, 并利用已建立的 Gal4/UAS 表达系统来研究细胞间的相互作用。

多色 FlpOut 技术在果蝇中的应用研究胶质细胞的高分辨率单细胞形态学

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Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

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A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here ...

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

一种新的方法，以评估在帕金森病大鼠脑深部电刺激效应的运动结果：楼梯和缸测试

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to ...

Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat

可逆冷却诱导 Deactivations 对行走猫的障碍记忆的皮层贡献研究

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For ...

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

在果蝇中结合定量食物摄入量测定和强迫激活神经元研究食欲

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, ...

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

一种自动测定果蝇在空间和时间温度变化中的性能的方法

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific ...

Assessing the Coherence of Parents' Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

使用五分钟言语样本程序评估家长对孩子的简短叙述的一致性

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with ...

Radiotracer Administration for High Temporal Resolution Positron Emission Tomography of the Human Brain: Application to FDG-fPET

人脑高时态分辨率正态发射断层扫描的放射跟踪器管理：在FDG-fPET中的应用

Functional positron emission tomography (fPET) provides a method to track molecular targets in the human brain. With a radioactively-labelled glucose ...