Name: quantifying synapses: an immunocytochemistry-based assay to quantify synapse number
Uploaded: 2010-11-16T17:30:00
Description: Watch this Scientific Journal Video about Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number at JoVE.com

Puncta分析仪插入图像J的巴里Wark（地址：Physion咨询）写在本A.巴雷斯实验室（斯坦福大学）。 资金; <ul><li> Alfred P. Sloan基金会</li><li>以斯帖A.和约瑟夫Klingenstein基金公司</li><li>广泛的生物医学研究基金会</li><li>治疗亨廷顿氏病倡议</li></ul>

准备的解决方案： <ol><li>抗体缓冲液： <ul><li> 150 mM氯化钠</li><li> 50 mM的三基（费舍尔​​，猫编号：BP152 - 5，50毫米） - 1.21克</li><li> 1％BSA（Sigma公司，没有猫：A2153，1％） - 2.0克</li><li> 100毫米L -赖氨酸（西格玛，猫编号：L - 1137，100毫米） - 3.65克</li><li>调整pH值至7.4 </li><li> 0.04％叠氮</li><li>音量调整到200毫升，用蒸馏水H 2 O </li><li>通过0.22μm的过滤器的过滤器（Millipore公司，卡?...

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	<li>Barres, B. A., Silverstein, B. E., Corey, D. P., Chun, L. L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological,+morphological,+and+electrophysiological+variation+among+retinal+ganglion+cells+purified+by+panning.">Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning.</a> Neuron. 1, 791-803 (1988).</li><li>Meyer-Franke, A., Kaplan, M. R., Pfrieger, F. W., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+signaling+interactions+that+promote+the+survival+and+growth+of+developing+retinal+ganglion+cells+in+culture.">Characterization of the signaling interactions that promote the survival and growth of developing retinal ganglion cells in culture.</a> Neuron. 15, 805-819 (1995).</li><li>Eroglu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gabapentin+receptor+alpha2delta-1+is+a+neuronal+thrombospondin+receptor+responsible+for+excitatory+CNS+synaptogenesis.">Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis.</a> Cell. 139, 380-392 (2009).</li><li>Pfrieger, F. W., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+efficacy+enhanced+by+glial+cells+in+vitro.">Synaptic efficacy enhanced by glial cells in vitro.</a> Science. 277, 1684-1687 .</li><li>Ullian, E. M., Sapperstein, S. K., Christopherson, K. S., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+synapse+number+by+glia.">Control of synapse number by glia.</a> Science. 291, 657-661 (2001).</li><li>Christopherson, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombospondins+are+astrocyte-secreted+proteins+that+promote+CNS+synaptogenesis.">Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis.</a> Cell. 120, 421-433 (2005).</li><li>Godement, P., Salaun, J., Imbert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prenatal+and+postnatal+development+of+retinocollicular+projections+in+the+mouse.">Prenatal and postnatal development of retinocollicular projections in the mouse.</a> J. Comp. Neurol. 230, 552-575 (1985).</li><li>Schmidt, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+retinotopic+connections:+selective+stabilization+by+an+activity-dependent+mechanism.">Formation of retinotopic connections: selective stabilization by an activity-dependent mechanism.</a> Cell. Mol. Neurobiol. 5, 65-84 (1985).</li><li>Sachs, G. M., Jacobson, M., Caviness, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+changes+in+arborization+patterns+of+murine+retinocollicular+axons.">Postnatal changes in arborization patterns of murine retinocollicular axons.</a> J. Comp. Neurol. 246, 395-408 (1986).</li><li>Elmariah, S. B., Oh, E. J., Hughes, E. G., Balice-Gordon, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+regulate+inhibitory+synapse+formation+via+Trk-mediated+modulation+of+postsynaptic+GABAA+receptors.">Astrocytes regulate inhibitory synapse formation via Trk-mediated modulation of postsynaptic GABAA receptors.</a> J. Neurosci. 25, 3638-3650 (2005).</li><li>Hughes, E. G., Elmariah, S. B., Balice-Gordon, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+secreted+proteins+selectively+increase+hippocampal+GABAergic+length,+branching+and+synaptogenesis.">Astrocyte secreted proteins selectively increase hippocampal GABAergic length, branching and synaptogenesis.</a> Moll. Cell. Neurosci. 43, 136-145 (2010).</li></ol>

上文所述突触检测是基于在我们的实验目标，其中主要集中于视网膜神经节细胞兴奋的预测中，无论是在纯化的文化，或在大脑部分。我们提供了一个参考表中列出的标签（见表1）兴奋性突触的抗体，工作。 这种突触的检测，可适应量化任何神经元的人口或任何其他的突触亚型，这是有选择性的前和突触后标记的突触数量。例如，突触实验描述在这里可以应用到研究的GABA - ...

<table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> 抗原 </td><td> 物种 </td><td> 单克隆/多克隆 </td><td> 供应商 </td><td> 产品编号 </td><td> 稀释 </td><td> 文化工程 </td><td> 可在第 </td></tr><tr><td rowspan="10">突触前</td><td>突触</td><td>兔</td><td>多克隆</td><td>突触系统</td><td> 106004 </td><td> 1:750 </td><td> Ÿ </td><td> ND </td></tr><tr><td>突触</td><td>鼠标</td><td>单克隆抗体</td><td>突触系统</td><td> 106001 </td><td> 1:500 </td><td> Ÿ </td><td> Ÿ </td></tr><tr><td>巴松管</td><td>鼠标</td><td>单克隆抗体</td><td>检测设计</td><td> VAM - PS003F </td><td> 1:500 </td><td> Ÿ </td><td> Ÿ </td></tr><tr><td>巴松管</td><td>豚鼠</td><td>多克隆</td><td>突触系统</td><td> 141004 </td><td> 1:1000 </td><td> Ÿ </td><td> ND </td></tr><tr><td>突触结合蛋白1 </td><td>兔</td><td>多克隆</td><td>突触系统</td><td> 105002 </td><td> 1:750 </td><td> Ÿ </td><td> ñ </td></tr><tr><td> Synaptobrevin 2 （C1.69.1） </td><td>鼠标</td><td>单克隆抗体</td><td>突触系统</td><td> 104211 </td><td> 1:500 </td><td> Ÿ </td><td> Ÿ </td></tr><tr><td>突触 （C1.7.2） </td><td>鼠标</td><td>单克隆抗体</td><td>突触系统</td><td> 101011 </td><td> 1:500 </td><td> Ÿ </td><td> Ÿ </td></tr><tr><td> VGlut1 </td><td>鼠标</td><td>单克隆抗体</td><td> Millipore公司</td><td> MAB5502 </td><td> 1:2500 </td><td> ñ </td><td> Ÿ </td></tr><tr><td> VGlut1 </td><td>豚鼠</td><td>多克隆</td><td> Millipore公司</td><td> AB5905 </td><td> 1:2500 </td><td> ñ </td><td> Ÿ </td></tr><tr><td> VGlut2 </td><td>豚鼠</td><td>多克隆</td><td> Millipore公司</td><td> AB2251 </td><td> 1:2500 </td><td> ñ </td><td> Ÿ </td></tr><tr><td rowspan="6">突触后</td><td> PSD - 95 （6G6 - 1C9克隆） </td><td>鼠标</td><td>单克隆抗体</td><td>亲和生物试剂</td><td> MA1 - 045 </td><td> 1:750 </td><td> Ÿ </td><td> ñ </td></tr><tr><td> PSD - 95 </td><td>兔</td><td>多克隆</td><td> Zymed公司</td><td> 51-6900 </td><td> 1:500 </td><td> ñ </td><td> Ÿ </td></tr><tr><td>荷马</td><td>鼠标</td><td>单克隆抗体</td><td>突触系统</td><td> 160011 </td><td> 1:500 </td><td> Ÿ </td><td> ND </td></tr><tr><td>荷马</td><td>大鼠</td><td>多克隆</td><td> Millipore公司</td><td> AB5875 </td><td> 1:500 </td><td> Ÿ </td><td> Ÿ </td></tr><tr><td> Gephyrin </td><td>兔</td><td>多克隆</td><td>突触系统</td><td> 147003 </td><td> 1:500 </td><td> Ÿ </td><td> Ÿ </td></tr><tr><td> Gephyrin </td><td>鼠标</td><td>单克隆抗体</td><td>突触系统</td><td> 147 </td><td> 1:200 </td><td> Ÿ </td><td> Ÿ </td></tr></tbody></table> 表1：好，我们已经成功地利用我们的突触检测前和突触后的标记列表的例子。要知道，这不是一个详尽的列表所有可用的标记。 Y =是，N =否，ND =未确定。 <table border="1" cellpadding="0" cellspacing="0"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 猫。号 </td></tr><tr><td> PBS </td><td> Invitrogen公司</td><td> 20012-027 </td></tr><tr><td>聚- D -赖氨酸</td><td>西格玛</td><td> P6407 </td></tr><tr><td>层粘连蛋白</td><td> Cultrex </td><td> 3400-010-01 </td></tr><tr><td> TRITON X - 100 </td><td>罗氏诊断有限公司</td><td> 9002-93-1 </td></tr><tr><td>正常山羊血清</td><td> Gibco公司</td><td> 16210 </td></tr><tr><td> VectaShield用DAPI </td><td>媒介实验室</td><td> H - 1200 </td></tr><tr><td>华侨城</td><td>组织Tek的</td><td> 4583 </td></tr><tr><td>三基地（50毫米） </td><td>费舍尔</td><td> BP152 - 5 </td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> A2153 </td></tr><tr><td> L -赖氨酸</td><td>西格玛</td><td> L - 1137 </td></tr><tr><td> 16％PFA解决方案</td><td>电子显微镜科学</td><td> 15711 </td></tr><tr><td>颗粒煤灰</td><td>电子显微镜科学</td><td> 19210 </td></tr><tr><td> 24孔培养板</td><td>鹘</td><td> 35-3047 </td></tr><tr><td>羊抗鼠Alexa的共轭抗体</td><td> Invitrogen公司</td><td> --- </td></tr><tr><td> 供应 </td><td> 公司 </td><td> 猫。号 </td></tr><tr><td> 12MM，0号盖玻片</td><td>卡尔赫克特有限公司</td><td> 1105209 </td></tr><tr><td> 1.5玻璃盖玻片（片） </td><td> VWR科学</td><td> 48393241 </td></tr><tr><td>载玻片</td><td> VWR科学</td><td> 48311-703 </td></tr></tbody></table>

quantifying synapses: an immunocytochemistry-based assay to quantify synapse number

在神经科学中最重要的目标之一是要了解分子线索指示突触形成的早期阶段。因此，它已成为当务之急，制定客观的方法来量化突触连接的变化。从样品固定开始，这个协议的细节如何量化无论是在分离的神经元文化和在脑切片，用免疫细胞化学突触的数量。使用车厢的特异性抗体，我们的标签突触前终端以及突触后专业化的网站。我们定义这些标记所产生的信号的共定位点之间的突触。这些colocalizations量化Puncta分析器（巴里Wark，可根据要求，c.eroglu @ cellbio.duke.edu书面）使用ImageJ分析软件平台下的一个插件。在本议定书中所描述的突触检测可应用于任何神经组织或文化的准备，你有选择性的前和突触后标记。这突触法是一种宝贵的的工具，可以被广泛利用在突触发展的研究。

Watch this Scientific Journal Video about Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number at JoVE.com

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

该协议的细节，如何量化，在分离神经元的文化和突触数量在脑切片，用免疫细胞化学。使用车厢的特异性抗体，我们的标签突触前终端以及突触后专业化的网站。我们定义这些标记所产生的信号的共定位点之间的突触。

突触量化：基于一个免疫细胞化学检测，以量化突触数量

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

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