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1.解决方案用于脑解剖和免疫荧光染色<ol><li>解剖成人飞脑在人造脑脊髓液（ACSF）:119 mM氯化钠，26.2毫碳酸氢钠 ，2.5mM的氯化钾，1mM的的NaH 2 PO 4，1.3毫氯化镁 ，和10mM葡萄糖。在使用前，气体用5％ 的 CO 2/95％O 2，10的脑脊液- 15分钟，穗带的2.5mM的CaCl 2。通过一个0.22微米的膜过滤器灭菌脑脊液溶液。 注:储存脑脊液在4℃，以防止细菌污染。飞大脑也可以在磷酸盐缓冲盐水（PBS）溶液解剖，虽然对解剖大脑的最终成像质量两个夹层溶液的作用详细的比较没有被执行。 </li><li>使用在1×PBS中制备的固定剂溶液，其通常等分并储存在-20℃的4％多聚甲醛（PFA）。 CautioN:PFA是有毒和腐蚀性，应小心处理。 </li><li>使用1X PBS从大脑和最后的清洗步骤后冲洗PFA，解剖大脑的免疫荧光染色过程中。从10倍的PBS原液制备1×PBS中（1.37 M氯化钠，27毫米氯化钾，100mM的磷酸氢二钠，和18毫KH 2 PO 4）。 </li><li>解剖脑的免疫荧光染色期间在1×PBS中（1×PBT）的所有随后的洗涤步骤使用0.3％吐温-20。 </li></ol> 2.成年果蝇首脑显微解剖 注意:解剖过程在图1中示出。 <ol><li>用CO 2麻醉成年果蝇。 5秒 - 使用镊子，拿起一只苍蝇，并简要由70％乙醇浸泡3就其脱蜡角质层。这确保了沉浸在脑脊液或1X PBS夹层溶液时没有气泡附着于飞角质层。 </li><li>下在那个不会被手被阻塞的角度解剖与光源的显微镜，设置物镜达到飞（1.2X倍率）一个明确的说法。使用镊子用非优势手来固定动物和浸入飞入其腹部朝上， 如图1A中的冷夹层溶液。 </li><li>保持镊子在160 - 170°角相对于夹层板，同时施加一个小的力到飞行的腹部（示出为图1A中的箭头）。这一步将固定飞行，并迫使它由15向后移动头部 - 向上延伸长鼻25°一会儿。在此过程中，表皮的柔软和半透明区域，长鼻下方，应当是显而易见的。增加放大倍率和调整焦平面到这一地区。 注意:不要拿着镊子得太紧，而只是足够牢固，能够稳定飞。用力过猛将导致内部器官破裂到溶液中。 </li><li>使用优势手按解剖镊，以便其尖端封闭的，这将产生对钳子的前端温和耐力，以便从保持手压被释放时的镊子将弹开。钳子垂直定位到保持飞镊子， 如图1B所示 。 <ol><li>皮尔斯解剖钳通过长鼻下方的角质层柔软和半透明区域闭合的提示，由图1B中的红色箭头所示。不穿透钳子太深它接触大脑是重要的。大脑正确的应该变得可见。保持飞与非优势手稳定的把握。 </li></ol></li><li>很快但稳定，靠自身的力量释放解剖钳提示，并依靠势头从这个版本到撕去Ť他的外骨骼周围的苍蝇头。一个完整的脑正确应该成为可见（白色组织立即在图1C的底部钳子的前端以上）跟随的假想线引导释放钳子提示（如在图1C中所示的向外红色箭头示出）的动能。这将温和地去除骨骼，大部分来自大脑的脑相关的眼睛和气管。适当的时机和力施加到打开外骨骼将取决于研究者的经验。 注:这是在解剖最关键的一步。它依赖于从开口钳的动量产生的自然力量，同时留下露出的脑仍附着在身体的其他部分以除去外骨骼是重要的。 </li><li>使用优势手的镊子小心地将残余附件组织，如出现白色纤维结构的气管remaini纳克附着到大脑。要小心，不要拉扯或损伤大脑正确的。 </li></ol> 3.大脑的免疫荧光染色<ol><li>固定在约1毫升4％PFA进行40与它们相关的躯干的解剖脑样品 - 60分钟。 注意:对于这个和随后的步骤，保持上一章动器的管或板的样品在溶液中适当地混合的样品。 </li><li>吹打解决方案上下3次用1毫升1X PBS的冲洗。小心不要吸出的大脑了。 </li><li>用1×PBT洗5次，每次5分钟。保持对章动器样品孵化过程中。 </li><li>一抗孵育适当的稀释O / N在4℃。 </li><li>第二天，除去第一抗体，并用1×PBT洗样品5次。离开样品5 - 在1倍的PBT溶液10分钟，每次洗涤之间。保持对章动器样品孵化过程中。 </li><li>孵育洗样中所建议的稀释和培养时间适当的二级抗体。 </li><li>用1X PBT洗样品六次每次冲洗之间的温育10分钟。 本步骤是从样品中除去非特异性结合的第二抗体是至关重要的。 </li><li>孵育以1:在1×PBT溶液的1毫克/毫升4&#39;，6&#39;-二脒基-2-苯基吲哚（DAPI）1:10,000稀释30分钟。 DAPI是结合DNA的AT区，可用于揭示脑的整体形态的DNA染料。 </li><li>用1X PBS冲洗三次。 </li><li>转移样品解剖菜，然后使用镊子解剖盘分开尸体的大脑。 </li><li>后染色和洗涤步骤，装入在盖玻片两者之间的大脑，并放置在盖玻片上的安装滑道的顶部。以这种方式，所述脑样品可以容易地翻转使大脑的两侧可具有类似的信号强度进行成像。 <strong&gt;注意:安装脑盖玻片上，而不是直接在安装滑道使得样品的方向翻转自如。安装工序是在下面更详细说明。 <ol><li>放置在安装滑道的顶部的18×18mm的盖玻片。加宽枪头用刀片的开口，并用它来在1×PBS溶液转移大脑到18×18mm的盖玻片。 </li><li>放置的大脑上盖玻片的中间，除去用细枪头大脑周围液体，然后添加另外20 - 抗褪色封固剂的40微升（0.2％（重量/体积）正丙基99.5没食子酸％ACS级甘油）在大脑。 注:固定介质中加入的量取决于研究大脑的数量。 </li><li>轻轻散开的大脑，而向外移位粘性安装介质。放置在脑的顶部的第二18×18mm的盖玻片通过首先从一侧降低滑动直到其与接触安装滑的表面上，然后慢慢降低的另一侧，以尽量减少气泡脑接触。 注意:没有必要使用垫片在两个盖玻片之间，作为安装培养基的体积和大脑应提供足够的两个盖玻片分开的空间。 </li><li>允许的幻灯片落户。除去过量的液体，从用实验室组织卡瓦两侧出来。 </li><li>为了防止安装的大脑盖玻片在运输过程中脱落的安装滑轨，用一小块胶带将盖玻片安装到固定的幻灯片。 注意:脑安装在之间的幻灯片可以立即被成像或在没有荧光信号的显著损失4℃长达一个星期保存下来，而不需要使用任何密封剂密封的幻灯片。对于染色的大脑长期保存，这两个滑动的侧可被密封瓦特第i指甲油，并存储在-20℃冰箱，虽然染色的大脑可能会失去它们的信号在一段时间。这是不推荐的。 </li></ol></li><li>使用适当的复合荧光显微镜或共聚焦显微镜图像的大脑。 <ol><li>首先，从大脑更靠近物镜的一侧获取图像，然后小心地翻转的盖玻片有夹在中间的试样。这个步骤有利于获得相同的大脑的另一侧的图像。 </li><li>使用一个直立化合物荧光显微镜和20X物镜到图像全脑（ 图2中的例子）和40X物镜到大脑的图像较小的区域，如在配对前内侧（PAM）簇区多巴胺神经元（示例在图3中）。 </li></ol></li></ol>

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The authors have nothing to disclose.

在使用成年果蝇脑研究人类脑疾病，神经元的电路，和更高的脑功能的兴趣日益增加，需要开发简单和快速的方法，以获得完整的飞大脑对于整个贴装分析，这是适用于大尤其重要比例基于大脑的屏幕。我们的方法提供了一个简单，易于学习的方法来剖析出一只苍蝇头（通常在不到10秒的经验）有保存完好的形态被清除大部分相关的组织。作为解剖大脑仍附着到飞体的其余部分，它们通常?...

模式生物果蝇 ，俗称实蝇，长期以来一直重视其优雅的基因工具，短生育时间和高度保守的分子和细胞通路。果蝇已成功地用于解剖基本信号通路，多细胞生物体的图案化的机制，以及神经元发育，功能的基本机制，和疾病1,2-。在细胞标记和成像技术的最新进展，果蝇大脑已经成为神经回路的精细定位，并在解剖的高级脑功能，例如学习，记忆，和昼夜节律1,3的分子和细胞基础特别强大， 4,5,6,7,8。 果蝇系统的一个特别的优点是它的相对小的尺寸，允许使用常规化合物或共焦显微镜的大脑整个安装制备和检查。氏取值特征使神经元电路的详细解剖和功能分析，或甚至一个单一的神经元，在细胞和亚细胞水平，在全脑组织的范围内，从而在整个内提供所研究的受试者的两个整体视图及其精确几何脑。然而，考虑到大脑的相当小尺寸，它也呈现在一个成年果蝇有效解剖一个完整的脑组织从保护外骨骼头壳体的技术挑战。各种有效和相对简单的解剖的方法已详细描述，其通常涉及仔细和分步除去头部壳体的和相关的组织，包括眼睛，气管，和脂肪从大脑适当9,10，这些显微解剖的方法经常放置在解剖钳的质量相当严格的要求，依托细以及对齐提示，可以很容易损坏钳。此外，作为解剖大脑往往分隔符从身体的其余编，大脑可以容易地期间，因为它们的小尺寸和它们在处理缓冲器透明度的后续染色和洗涤过程丢失。在这里，我们描述了，保持连接到躯干的解剖大脑成人大脑相对简单和容易学习，一步夹层协议。解剖过程中往往容易清除了大部分脑相关组织如眼睛和气管，并减少了质量好的解剖钳的需求。 此外，荧光化合物显微镜或共焦显微镜下成像大脑时，大脑的一侧即来自荧光光源远经常产生一个较弱的信号和较少的清晰图像，由于整个贴装脑的厚度。在这里，我们还描述了一个简单的安装方法，其允许大脑样品容易翻转，使大脑两侧方便成像相似的信号的IntensiTY和质量。 作为证明的概念对于本方法的应用，研究成人脑，我们进一步研究多巴胺神经元的用w 1118蝇脑中的存在;被经常用作用于产生转基因苍蝇，在许多果蝇研究野生型对照的亲本系基因型。

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	<tbody>
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			<td height="54" style="height:54px;width:573px;">w*;&#160;park&#916;21/TM3,&#160;P{GAL4-Kr.C}DC2,&#160;P{UAS-GFP.S65T}DC10,&#160;Sb1</td>
			<td style="width:311px;">Bloomington Drosophila Stock Center</td>
			<td style="width:151px;">51652</td>
			<td style="width:287px;">Balancer was switched to TM6B</td>
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		<tr height="39">
			<td height="39" style="height:39px;">PBac{WH}parkf01950</td>
			<td>Exelixis at Harvard Medical School</td>
			<td>f01950</td>
			<td>Balancer was switched to TM6C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">NaCl</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>S640-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:573px;">Sodium Bicarbonate (NaHCO3)&#160;</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td style="width:151px;">02-003-990</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Potassium Chloride (KCl)</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>BP366-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:573px;">Sodium phosphate, monobasic monohydrate (NaHCO3)</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>02-004-198</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Magnesium Chloride (MgCl2)</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>02-003-265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">D-Sorbitol</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>S1876-500G</td>
			<td>Replaces glucose</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:573px;">Calcium chloride dihydrate (CaCl2)</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>C5670-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EMD Millipore&#160;Durapore PVDF Membrane Filters: Hydrophilic: 0.22&#181; Pore Size</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>GVWP14250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Formalin Solution, 10% (Histological)</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>SF98-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Potassium Phosphate, Dibasic, Powder, Ultrapure Bioreagent</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>02-003-823</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Tween 20</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Excelta Precision Tweezers with Very Fine Points</td>
			<td style="width:311px;">Fisher Scientific</td>
			<td>17-456-055</td>
			<td>Protocol does not require very fine points.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Anti-Tyrosine Hydroxylase Antibody</td>
			<td>Pel-Freez Biologicals</td>
			<td style="width:151px;">P40101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Rat-Elav-7E8A10 anti-elav</td>
			<td>The Developmental Studies Hybridoma Bank</td>
			<td>Clone 7E8A10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 647 conjugate</td>
			<td style="width:311px;">ThermoFisher Scientific</td>
			<td style="width:151px;">A-21247</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate</td>
			<td style="width:311px;">ThermoFisher Scientific</td>
			<td style="width:151px;">A-11037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">DAPI Solution (1 mg/mL)</td>
			<td style="width:311px;">ThermoFisher Scientific</td>
			<td style="width:151px;">62248</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propyl gallate powder</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>P3130-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Glycerol ACS reagent, &#8805;99.5%</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>G7893-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss Axioimager Z1</td>
			<td style="width:311px;">Zeiss</td>
			<td style="width:151px;">Quote</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Zeiss Apotome.2</td>
			<td style="width:311px;">Zeiss</td>
			<td style="width:151px;">Quote</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:573px;">Zen lite software</td>
			<td style="width:311px;"></td>
			<td style="width:151px;">Quote</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple one-step dissection protocol for whole-mount preparation of adult drosophila brains

有在使用果蝇到人脑退行性疾病的模型，在成人大脑映射神经设计电路，和研究的高级脑功能的分子和细胞基础的兴趣日益增加。成人大脑的整体卡口制剂保存完好的形态是这样的全脑为基础的研究关键的，但也可以是技术挑战性和耗时。这个协议描述了成年果蝇头部在不到10秒的一个易于了解，一步法解剖的方法，同时保持附连到身体的其他部分的完整的脑，以方便后续处理步骤。该过程有助于消除最通常与可以与稍后成像步骤干涉大脑相关的眼睛和气管组织的，并且也对所述解剖钳的质量需求较少。此外，我们描述一个简单的方法，可用于在盖玻片安装的脑样品的方便翻转，这是用于成像的B的两侧重要降雨具有类似信号强度和质量。作为协议的一个例子，我们在野生成年大脑多巴胺能呈现（DA）神经元的分析（ 附1118）苍蝇。解剖方法的疗效高，使其成为在果蝇中大型为主脑的成人研究特别有用。

图1示出了用于成人脑解剖主程序中，如上所述图2和3是3日龄WT代表性图像（基因型:W 1118）成人飞大脑，其被costained用抗酪氨酸羟化酶的抗体（TH ，用红色的图2和白色图3），一个标记通常用于标记多巴胺神经元11中，除了将DNA染料DAPI标记所有所有分化的神经元细胞的细胞核和针对ELAV蛋...

Watch this Scientific Journal Video about A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains at JoVE.com

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

成年果蝇大脑是研究神经回路，大脑高级功能和复杂性疾病的宝贵制度。一种有效的方法来剖析来自小苍蝇头全脑组织将促进脑为基础的研究。在这里，我们描述了一个简单的一步解剖成人的大脑保存完好的形态协议。

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

a-simple-one-step-dissection-protocol-for-whole-mount-preparation

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

27.0K 次观看.  The University of Texas Health Science Center at Houston. 成年果蝇大脑是研究神经回路，大脑高级功能和复杂性疾病的宝贵制度。一种有效的方法来剖析来自小苍蝇头全脑组织将促进脑为基础的研究。在这里，我们描述了一个简单的一步解剖成人的大脑保存完好的形态协议。 

视频: A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

在全安装的胚胎神经系统的可视化果蝇胚胎

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

科研

神经科学

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

解剖鼠标眼的视网膜色素上皮的全山

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

单果蝇 Ommatidium解剖和影像

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled samples with little to no tissue damage enables us to analyze cell morphology and to compare individual samples to each other, hence allowing the identification of mutant anomalies.
In the demonstrated dissection method the nervous system remains mostly inside the adult fly. Through a dorsal incision, the abdomen and thorax are opened and most of the internal organs are removed. Only the dorsal side of the ventral nerve cord (VNC) and the cervical connective 
(CvC) containing the big axons of the giant fibers (GFs)1 are exposed, while the brain containing the GF cell body and dendrites remains2 in the 
intact head. In this preparation most nerves of the VNC should remain attached to their muscles. 
Following the dissection, the intracellular filling of the giant fiber (GF) with a fluorescent dye is demonstrated. In the CvC the GF axons are located at the dorsal surface and thus can be easily visualized under a microscope with differential interference contrast (DIC) optics. This allows the injection of the GF axons with dye at this site to label the entire GF including the axons and their terminals in the VNC. This method results in reliable and strong staining of the GFs allowing the neurons to be imaged immediately after filling with an epifluorescent microscope. Alternatively, the fluorescent signal can be enhanced using standard immunohistochemistry procedures3 suitable for high resolution confocal microscopy.

Whole Mount Preparation of the Adult Drosophila Ventral Nerve Cord for Giant Fiber Dye Injection

An in vivo dissection of the adult Drosophila ventral nerve cord (VNC) is demonstrated. This particular dissection method causes little damage to the VNC allowing the subsequent labeling of the giant fiber neurons with fluorescent dye for high resolution imaging.

成人整装准备果蝇巨纤维染料注射腹侧神经索

To analyze the axonal and dendritic morphology of neurons, it is essential to obtain accurate labeling of neuronal structures. Preparing well labeled ...

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

DII的标签DRG神经元的研究轴突分支在小鼠胚胎脊髓整装准备

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

幼虫，蛹和成人的解剖和免疫组化<em&gt;果蝇</em&gt;视网膜

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

全山神经肌肉交界分析的 横向解剖阿布多米尼斯 肌肉

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

全山解剖和成年小鼠耳蜗的免疫

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

一个简单的一步解剖协议成人全安装准备

摘要

探索更多视频

此视频中的章节

在全安装的胚胎神经系统的可视化果蝇胚胎

解剖鼠标眼的视网膜色素上皮的全山

单果蝇 Ommatidium解剖和影像

成人整装准备果蝇巨纤维染料注射腹侧神经索

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

DII的标签DRG神经元的研究轴突分支在小鼠胚胎脊髓整装准备

幼虫，蛹和成人的解剖和免疫组化

全山神经肌肉交界分析的横向解剖阿布多米尼斯肌肉

嗅觉受体神经元在全山免疫标记

全山解剖和成年小鼠耳蜗的免疫

一个简单的一步解剖协议成人全安装准备

摘要

探索更多视频

此视频中的章节

在全安装的胚胎神经系统的可视化果蝇胚胎

解剖鼠标眼的视网膜色素上皮的全山

单果蝇 Ommatidium解剖和影像

成人整装准备果蝇巨纤维染料注射腹侧神经索

重复性小鼠坐骨神经挤压和再生的后续评估整装肌肉分析

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