Name: sequential photo-bleaching to delineate single schwann cells at the neuromuscular junction
Uploaded: 2013-01-11T13:00:00
Description: Watch this Scientific Journal Video about Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction at JoVE.com

我们想感谢曼努埃拉·布达克，Ljiljana Marinkovi和克里斯蒂娜Wullimann的，为优秀的技术援助。我们感谢W.麦克林提供PLP-GFP小鼠。 TM高级研究所（慕尼黑工业大学），德意志研究联合会（DFG Sonderforschungsbereich SFB 596），亚历山大冯洪堡基金会支持，由国家提供资金的机构（的“Bundesministerium献给教化和Forschung” ）ERA-净神经元“iPSoALS”和“2 - 光子成像”中的帧的。 TM和MB支持由集成蛋白质科学中心（慕尼黑）。 PM由研究生院的慕尼黑工业大学（TUM-GS）的支持。

1。三角Sterni外植体（ 图1） 定时：15分钟。 <ol><li>准备手术器械：2双钳，剪刀，春风似剪刀1对，1个组织培养皿中（10厘米）。预鼓泡（至少15分钟）冷却的（4°C）的林格氏溶液，5％CO 2/95％O 2。填充15厘米的冰和组织培养皿中与金属板覆盖。准备解剖显微镜下的清扫范围内，放置在15-cm培养皿中，用冰冷为了冷却剥离时的外植体。 </li><li>致死麻醉小鼠?...

<ol>
	<li>Feng, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+in+transgenic+mice+expressing+multiple+spectral+variants+of+GFP.">Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.</a> Neuron. 28, 41-51 (2000).</li><li>Mallon, B. S., Shick, H. E., Kidd, G. J., Macklin, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolipid+promoter+activity+distinguishes+two+populations+of+NG2-positive+cells+throughout+neonatal+cortical+development.">Proteolipid promoter activity distinguishes two populations of NG2-positive cells throughout neonatal cortical development.</a> J. Neurosci. 22, 876-885 (2002).</li><li>Brill, M. S., Lichtman, J. W., Thompson, W., Zuo, Y., Misgeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+constraints+dictate+glial+territories+at+murine+neuromuscular+junctions.">Spatial constraints dictate glial territories at murine neuromuscular junctions.</a> The Journal of cell biology. 195, 293-305 (2011).</li><li>Livet, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+strategies+for+combinatorial+expression+of+fluorescent+proteins+in+the+nervous+system.">Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.</a> Nature. 450, 56-62 (2007).</li><li>Thompson, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+mice+with+photoswitchable+GFP+in+Schwann+cells.">Transgenic mice with photoswitchable GFP in Schwann cells.</a> Society for Neuroscience. Program 241, (2008).</li><li>Williams, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+development+of+outer+retinal+synapses+in+the+absence+of+glial+contact.">In vivo development of outer retinal synapses in the absence of glial contact.</a> J. Neurosci. 30, 11951-11961 (2010).</li><li>Kerschensteiner, M., Reuter, M. S., Lichtman, J. W., Misgeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+imaging+of+motor+axon+dynamics+in+murine+triangularis+sterni+explants.">Ex vivo imaging of motor axon dynamics in murine triangularis sterni explants.</a> Nat. Protoc. 3, 1645-1653 (2008).</li><li>McArdle, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advantages+of+the+triangularis+sterni+muscle+of+the+mouse+for+investigations+of+synaptic+phenomena.">Advantages of the triangularis sterni muscle of the mouse for investigations of synaptic phenomena.</a> J. Neurosci. Methods. 4, 109-115 (1981).</li><li>Marinkovi, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+deficits+and+degeneration+can+evolve+independently+in+mouse+models+of+amyotrophic+lateral+sclerosis.">Axonal transport deficits and degeneration can evolve independently in mouse models of amyotrophic lateral sclerosis.</a> Proceedings of the National Academy of Sciences of the United States of America. 109, 4296-4301 (2012).</li><li>Thevenaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE Trans Image Process. 7, 27-41 (1998).</li><li>Brill, M. S., Lichtman, J. W., Thompson, W., Zuo, Y., Misgeld, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+constraints+dictate+glial+territories+at+murine+neuromuscular+junctions.">Spatial constraints dictate glial territories at murine neuromuscular junctions.</a> The Journal of cell biology. 195, 293-305 (2011).</li><li>Zuo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+proteins+expressed+in+mouse+transgenic+lines+mark+subsets+of+glia,+neurons,+macrophages,+and+dendritic+cells+for+vital+examination.">Fluorescent proteins expressed in mouse transgenic lines mark subsets of glia, neurons, macrophages, and dendritic cells for vital examination.</a> J. Neurosci. 24, 10999-11009 (2004).</li></ol>

SC漂白这里介绍的方法很简单，速度快，通用性：（一）它使一个单一的基因-这是一个显着的优势相结合的方法， 例如需要额外的基因与疾病模型，激光共聚焦显微镜的基础上揭示单SC形态和动态。 （ii）该方法具有快速，从解剖，以固定它可以在半天的时间。 （ⅲ）的应用程序的数目是广泛的，因为它可以与其他方法，如细胞消融，成像的细胞器，电生理学或免疫组织化学结合。此外?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司名称（示例） </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td> 70％（体积/体积）的乙醇溶液</td><td>罗斯</td><td> T913.1 </td><td>在喷雾瓶</td></tr><tr><td>异氟醚</td><td> Forene，雅培</td><td></td><td>可以使用任何其他核准麻醉致死麻醉</td></tr><tr><td>转基因小鼠（PLP-GFP） </td><td></td><td></td><td>获得援引消息人士</td></tr><tr><td>解剖显微镜冷光照明</td><td>奥林巴斯SZ51 </td><td></td><td>配备了肖特KL 1500 LCD </td></tr><tr><td>镊子＃2 </td><td>精细科学工具</td><td> 11233-20 </td><td></td></tr><tr><td>镊子＃5 </td><td>精细科学工具</td><td> 11295-00 </td><td></td></tr><tr><td>剪刀，大型医疗</td><td>精细科学工具</td><td> 14108-09 </td><td></td></tr><tr><td>剪刀，小角度的春天</td><td>精细科学工具</td><td> 15033-09 </td><td></td></tr><tr><td>列直插式加热器</td><td>华纳仪器</td><td> SF-28 </td><td></td></tr><tr><td> 3.5厘米菜肴的加热圈</td><td>华纳仪器</td><td> 64-0110 DH-35 </td><td></td></tr><tr><td>两路温度控制系统</td><td>华纳仪器</td><td> TC-344B </td><td></td&gt; </tr><tr><td>灌流泵系统</td><td></td><td></td><td>定制的</td></tr><tr><td> 15 cm组织培养皿</td><td>萨尔斯塔特</td><td> 83.1803.003 </td><td>盛有冰和金属板覆盖</td></tr><tr><td> 10 cm组织培养皿</td><td>萨尔斯塔特</td><td> 83.1802.003 </td><td>用含氧林格溶液</td></tr><tr><td> 3.5 cm组织培养皿</td><td>萨尔斯塔特</td><td> 83.1800.003 </td><td>充满Sylgard的聚合物</td></tr><tr><td> Sylgard的聚合物</td><td>道康宁</td><td> Sylgard的184有机硅弹性体套件</td><td></td></tr><tr><td>激光共聚焦显微镜</td><td>奥林巴斯</td><td> FV1000 </td><td>用氩气激光</td></tr><tr><td> 50毫升的反应管</td><td>萨尔斯塔特</td><td> 62.547.254 PP </td><td></td></tr><tr><td>在PBS中的4％PFA </td><td>西格玛</td><td> P6148 </td><td></td></tr><tr><td>套管0.5毫米x 25毫米</td><td> Neolab </td><td> E-1510 </td><td></td></tr><tr><td>注射器1毫升</td><td> Neolab </td><td> E-1496 </td><td></td></tr><tr><td>突触素抗体</td><td> Invitrogen公司</td><td> 18-0130 </td><td>兔</td></tr><tr><td>二级抗体</td><td> Invitrogen公司</td><td> A-11012 </td><td> Alexa的594 </td></tr><tr><td> 24 - 孔板</td><td>萨尔斯塔特</td><td> 83.1836 </td><td></td></tr><tr><td> 0.01 M PBS </td><td>西格玛</td><td> P4417 </td><td></td></tr><tr><td>在PBS中的0.1M甘氨酸</td><td>罗斯</td><td> 3790.1 </td><td></td></tr><tr><td>盖玻片</td><td> Neolab </td><td> E-4132 </td><td></td></tr><tr><td>幻灯片</td><td> Neolab </td><td> E-4121 </td><td></td></tr><tr><td> Vectashield </td><td>矢量实验室</td><td> H-1000 </td><td></td></tr><tr><td> minutien引脚×10 </td><td>精细科学工具</td><td> 26002-20 </td><td>缩短至&lt;4毫米</td></tr><tr><td> α-银环蛇毒素结合网站594 </td><td> Invitrogen公司（Molecular Probes公司） </td><td> B-13423 </td><td></td></tr><tr><td></td><td></td><td></td><td> 封闭液 </td></tr><tr><td> 0。01 M PBS </td><td></td><td></td><td></td></tr><tr><td> 0.2％的Triton-X100 </td><td>罗斯</td><td> 3051.3 </td><td></td></tr><tr><td> 10％正常山羊血清</td><td> Abcam公司</td><td> ab7481 </td><td></td></tr><tr><td> 1％牛血清白蛋白</td><td>西格玛</td><td> A7030 </td><td></td></tr><tr><td></td><td></td><td></td><td> 林格 鼓泡通入95％O 2/5％CO 2的 </td></tr><tr><td> 125毫米的NaCl </td><td>西格玛</td><td> S7653 </td><td></td></tr><tr><td> 2.5毫米氯化钾</td><td>西格玛</td><td> P9333 </td><td></td></tr><tr><td> 1.25毫摩尔的NaH 2 PO 4的 </td><td>西格玛</td><td> S8282 </td><td></td></tr><tr><td> 26毫米碳酸氢钠 </td><td>西格玛</td><td> S6297 </td><td></td></tr><tr><td> 2毫米氯化钙 </td><td>西格玛</td><td> 21114 </td><td></td></tr><tr><td> 1毫摩尔MgCl 2 </td><td>西格玛</td><td> 63020 </td><td></td></tr><tr><td> 20mM葡萄糖</td><td>西格玛</td><td> G7528 </td><td></td></tr><tr><td></td><td></td><td></td><td> 表1中。具体的试剂和设备。 </td></tr></tbody></table>

sequential photo-bleaching to delineate single schwann cells at the neuromuscular junction

顺序光漂白在NMJ标记个别SC的，提供了一种非侵入性的方式。 NMJ是世界上最大的哺乳动物神经系统的突触，并担任指导模式研究突触的结构和功能。电机轴突在鼠标NMJs的终端形成与肌纤维的椒盐卷饼般的接​​触位点。公务员事务局局长电机轴突和终端的护套。在过去的几十年里，转基因小鼠已产生运动神经元和SC可视化，例如大鼠Thy1-XFP和PLP-GFP小鼠，分别。 沿着运动神经元轴突，髓鞘轴索SC的配置在非重叠的节间，分离由节点朗维埃，启用跳跃动作电位传播。相比之下，公务员事务局局长的突触终端是专门的神经胶质细胞，监督和促进神经传导，消化碎片和引导轴突再生。的NMJs盖好了半打非MYELinating端子SC的- ，但是，这些不能被单独用光学显微镜解决，因为它们是在直接膜接触3。 有几种方法可以单独可视化终端种姓。这些都不是完美的，虽然。例如，染料填充，单细胞染料填充微刺穿，需要摧毁了标记的细胞，然后再填充第二个。与随后的时间推移录音3，这是一个不兼容。多光谱“Brainbow”标签的判已经实现，通过使用组合的荧光蛋白的表达4。然而，该技术需要结合几个转基因和限制使用的启动子的表达模式。在未来，“照片切换的”蛋白质在SC的表达可能会对又一替代5。在这里，我们提出了连续的光漂白，漂白单细胞，和自己的形象得到减法。我们相信，这种方法-由于其易用性和多功能性-代表持久除了神经学家的技术调色板，特别是因为它可以在体内使用，转移到其他类型的细胞，解剖部位或物种6。 在下面的协议，我们详细的应用顺序漂白时间的推移和随后的共聚焦显微镜终端公务员事务局局长的三角sterni肌肉植。这本薄薄的，肤浅的，容易解剖神经肌肉准备7,8证明是有益的研究NMJ发展，生理和病理9。最后，我们解释了如何的三角sterni肌肉固定后准备执行相关的高解析度激光共聚焦成像，免疫组化或超微结构检查。

一个准备好成像菜的的三角sterni外植体的一个例子是在图1G所示。此外植体是特别适合用于成像NMJs 体外作为三角的肌肉仅由数层的肌肉纤维。这允许获得高清晰度的图像，从外植体来源于转基因小鼠系，亮点要么判（PLP-GFP 2）和/或轴突（Thy1肾炎大鼠 -XFP 1）。高品质成像的关键因素包括：（i）避免接触三角肌肉外植体的制备过程中，以防止肌肉?...

Watch this Scientific Journal Video about Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction at JoVE.com

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

可视化的单个细胞密集的组织，如终端雪旺氏细胞（SC）的神经肌肉接头（NMJs），是具有挑战性的。 “连续光漂白”可以划定单一的终端南海，例如在的三角sterni肌肉外植体，方便神经肌肉准备，连续的漂白可以结合时间推移成像<em&gt;事后</em&gt; immunostainings。

连续光漂白划定单雪旺细胞在神经肌肉接头处

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Research

JoVE Journal

Neuroscience

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

在活动区域​​的解剖和影像果蝇神经肌肉交界处

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

科研

神经科学

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

阈上的神经活动的单细胞和单穗的分辨率的光学的方法记录

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

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

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

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 ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

检测<em&gt;原位</em&gt;蛋白质 - 蛋白质复合物的<em&gt;果蝇</em&gt;幼虫神经肌肉接头使用接近结扎分析

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

神经肌肉接头处：测量突触大小，碎片和改变突触蛋白密度利用共聚焦荧光显微镜

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

为什么量化事项：表型的表征在<em&gt;果蝇</em&gt;幼虫神经肌肉接头

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Measuring Neuromuscular Junction Functionality

测量神经肌肉接头功能

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

从果蝇幼虫神经肌肉交界处的突触电流的焦 Macropatch 记录

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

在体内果蝇前运动神经末端的单分子跟踪

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...