作者感谢Teele Palumaa与数字的援助。从国家健康和医学研究委员会，澳大利亚资助计划资助1008046。

1。大鼠海马神经元培养<ol><li>准备盖玻片（硼硅玻璃）： <ol><li>在100％乙醇洗涤。 </li><li>在紫外光照射下空气干燥。 </li><li>大衣与聚-D-赖氨酸（0.5毫克/毫升的0.15M硼酸盐缓冲液，4℃过夜°C）。 </li><li>在第二天（培养的天）的PBS洗涤3次，然后涂以层粘连蛋白（2.5微克/毫升，天然小鼠层粘连蛋白）+ 5％体积/体积的热灭活的胎牛血清（FCS）的PBS稀释为2小时，在37°C。 </li></ol></li><li>解剖胚胎18天（E18）大鼠海马和收集到PBS含有钙和镁，在冰上冷却。注意：所有涉及动物实验方案已获墨尔本大学动物伦理委员会批准。 </li><li>根据制造商的指示准备从木瓜蛋白酶解离试剂盒的木瓜蛋白酶和DNA酶I的解决方案。 <ol><li>加入50微升的DNase I溶液至1ml木瓜蛋白酶溶液。 </li><li>注意：一旦首先制备的木瓜蛋白酶溶液和DNA酶I溶液可以分别分配到等分试样（0.5 ml和25微升等份为木瓜蛋白酶和DNA酶I，分别）并储存于-20℃直至需要。 </li></ol></li><li>去除尽可能多的PBS，尽量与在37℃下1毫升木瓜蛋白酶/ DNA酶I溶液15-20分钟孵育海马（来自胚胎的1升）。轻轻一抖管在孵化期的两倍，混合内容。 </li><li>直至细胞分散，得到很少，如果有的话用火焰抛光的硅化的巴斯德移液管10-15X磨碎的海马组织轻轻地（避免气泡的产生），未解离的组织块仍然存在。 </li><li>仔细层解离的细胞悬液在一个3毫升坐垫的4％w / v的牛血清白蛋白（BSA）的Hanks平衡盐溶液加添加剂（HBSS +，见第1.6.1节）。 <ol><li>由无搅拌溶解BSA在室温下准备这一步梯度液含有2mM的CaCl 2，1mM的MgSO 4干燥，4毫米的NaHCO 3和40mM的葡萄糖（HBSS +），然后无菌过滤并储存于4℃。环在HBSS </li></ol></li><li>离心机，XG 100，在台式离心机具有水平转子7分钟。 </li><li>去除上清小心不要吸细胞沉淀。 </li><li>轻轻悬浮沉淀的细胞用火焰抛光（硅氧烷）巴斯德吸管（或1毫升蓝枪头）在1ml完整的Neurobasal培养基（含2％B27，0.5 2mM L-谷氨酰胺补充有1％FCS）中。 </li><li>使用血球计数两个10微升等份（注：只计算相亮细胞作为“活的”细胞）。 </li><li>板对预先制备镀膜玻璃盖玻片（0.75-1×10 5 /18毫米盖玻片在12孔板）初级神经元。紧接在电镀前，吸从盖玻片上多余的层粘连蛋白/血清溶液，并替换为初级神经元培养基（见下文）。注：所需要的n需要盖玻片的茶色被预先确定为盖玻片制备当天开始之前，培养物（参见步骤1.1）。 </li><li>培养的大鼠原代E18海马神经元在体外 （DIV）的Neurobasal培养基，2％B27，0.5 2mM L-谷氨酰胺补充有1％FCS（热灭活的）长达21天。在7 DIV和此后每周进行半换液。抗有丝分裂氟脱氧尿苷/尿嘧啶核苷被添加在7 DIV; 1/1，000稀释的每个核苷10mM的股票），以防止神经胶质增生。 </li></ol> 2。抗体孵育与Live神经元<ol><li>在培养的第一周，神经元树突开发乔木和2-3周，神经元已经成熟到足以被接受突触6,7。 <ol><li>在选定的实验时间点/秒，适用于一抗一式三份含有盖玻片上小学胚胎神经元井。加抗体的等分试样直接加入的培养基在原代神经元为所需的最终稀释度注：在本例中，提出了针对该蛋白的重组分泌形式的兔多克隆抗血清，稀释1/500，加入2微升到1毫升培养培养基中公;一个离心10分钟，13,000 XG，RT可以合并之前，采取抗体等分，因为这会擦除任何颗粒物质，可以帮助降低背景。 </li><li>对于免疫前血清对照，添加免疫前血清的等效量（以相同的最终稀释度）。如果没有免疫前血清是可用的，加Neurobasal培养基或PBS的等效体积（无一抗控制）。另外，如果合适的初级抗体可识别该蛋白质的胞内区/秒，该抗体可以被添加到一个控制以及为了测试表面染色的特异性。 </li><li>返回到细胞培养孵化1-4小时（此潜伏期可以根据经验确定）。在我们的experimeNTS，所述抗体是存在的整个期间，虽然实验设计可以结合抗体的一个脉冲之后进行后洗出的另一潜伏期（一个脉冲追踪实验），以评估内化的时间进程。 可选的步骤：此温育可以进行在室温下或什至在冰上如果有必要减缓基底蛋白内化的速率。如果在非CO 2的受控环境中进行的，该介质应改为一个不碳酸氢盐添加抗体/抗血清之前进行缓冲。注意：成熟的神经元培养（&gt; 14天，特别是培养的小鼠胚胎神经元）不耐受全媒介的变化以及。 </li></ol></li><li>我们已经使用的孵育时间为1小时，2小时，并具有良好的效果4小时的这一次抗体内化步骤虽然最佳的时间将取决于大量的感兴趣的蛋白质，以及蛋白质运输中的CEL的动态根据研究LS。 注：代表结果中显示的双彩色图像的细胞培养箱获得1小时的潜伏期在37℃。 </li><li>温育所需的时间后，吸脱含有抗体的培​​养基。在室温下用PBS轻轻，但很快，一旦洗井。 </li></ol> 3。二次抗体应用到固定，Unpermeabilized细胞<ol><li>修复神经元在磷酸盐缓冲液pH 7.2 4％多聚甲醛，5分钟，在室温下注：为达到最佳效果，使用新鲜制备的固定剂;交替使用已经存储了在-20℃下和完全解冻修正，以便没有沉淀的多聚甲醛是显而易见。注意：多聚甲醛固定液应准备，并在通风橱中进行处理。 </li><li>从井（转移到通风柜液体废物容器）拆下固定，冲洗3次，用PBS。 <ol><li>可选步骤：如果需要，以节省抗体试剂，盖玻片可以从12孔板用镊子小心地取出并放在单元侧时，上封口膜的片材布置的一次性培养皿的底部上。 </li><li>解决方案可以被轻轻地吸移到盖玻片，使它们不解决水浸过的盖玻片的边缘上的封口膜完全覆盖。 </li><li>此技术适用于短期孵育（1-2小时），但是更长的孵育（ 例如过夜），应在湿润的腔室中进行。可替换地，盖玻片可以放回12孔板的过夜培养和足够体积的孔中应加入以确保盖玻片不干燥。 </li></ol></li><li>为30分钟，在室温下用在PBS中的5％BSA（：不添加洗涤剂在此阶段封闭溶液，因为它是重要的细胞不透注）块。 </li><li>为了继续进行DETEC前表面标记蛋白内化蛋白化，应用第一个荧光标记2°抗体的选择。 注意：在这里描述的例子中，初级抗血清对重组分泌的同种型4中提出兔（住宅内抗体）。因此，对于第一个二次抗体来检测的跨膜同种型的胞外区unpermeabilized神经元的表面上，我们用驴抗兔Dylight 649（在PBS中稀释含有5％BSA的1/200）。建议在离心机在使用前稀释的二级抗体溶液（10分钟，13,000×g离心，RT）。 </li><li>孵育的盖玻片为2小时，在室温下进行。 </li><li>洗盖玻片（井），2个5分钟，用PBS。 </li></ol> 4。阻断与过量的未标记2°抗体<ol><li>用未标记的2°抗体的高浓度（&gt; 0.1毫克/毫升）阻断unpermeabilized神经元。 <ol><li>未标记的2°抗体应该对物种得到提升，其中初级抗体升高（在这种情况下兔）温育在室温下过夜。 </li><li>此协议，AffiniPure F AB片段山羊抗兔IgG（H + L）中的用量为0.13毫克/毫升的浓度。 注：过夜培养被认为是作为一个较短的潜伏期（2小时），关键是不够的初级抗体，该抗体未完全被标记的二次抗体结合的完全阻塞。 </li></ol></li><li>洗盖玻片（井），2个5分钟，用PBS。 </li><li>在此之后封闭步骤，后固定细胞用磷酸盐缓冲液（pH7.2）中5分钟，在室温下4％多聚甲醛。去除固定液（固定液转移到通风柜液体废物容器）后，用PBS（2X）冲洗。 </li></ol> 5。第二次将荧光共轭2°抗体的通透性及应用<ol><li>通透并阻断细胞用含有0.1％的Triton-X-100在室温坦佩5％BSA的PBSrature 30分钟。 </li><li>除去封闭液（注意不要让盖玻片干不出来）。添加第二个荧光标记的2°抗体标记有不同的荧光团。 <ol><li>注意：它必须有可能从先前所用的1区分这种荧光团标记，这取决于在共聚焦显微镜中使用的激发/发射过滤器（见下文）。 </li><li>在这个协议提出的例子中，一个的Alexa Fluor 488 - 标记的驴抗兔2°抗体被用于（在PBS中的1/200，5％BSA和0.1％的Triton-X-100）。孵育2小时，在室温下再取出2°抗体溶液。 </li></ol></li><li>洗涤盖玻片3×5分钟，用PBS，最后用去离子水短暂洗涤。 </li></ol> 6。安装和成像<ol><li>贴装盖玻片载玻片上用含水安装含有中等抗淬灭（ 例如 VECTASHIELD）并晾干。存放在暗处，在4℃下运timal保存荧光信号强度。 </li><li>图片上的免疫染色共聚焦显微镜细胞。 <ol><li>合适的激发和发射滤波器，用于检测2荧光信号必须是可用的。 </li><li>如果不同的实验条件进行比较（例如，内化去极化8,9或控制条件下的速率），确保所有复制从各种条件盖玻片成像相同的图像采集参数。感兴趣的标准区域或从得到的图像中的泪点的属性（ 例如，数量，大小）的集成密度然后可以使用标准图像分析软件（ 例如 ，斐济/ ImageJ的，的Metamorph）进行测定。 </li></ol></li></ol>

<ol>
	<li>Lewis, T. L., Mao, T., Arnold, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+myosin+VI+in+the+localization+of+axonal+proteins.">A role for myosin VI in the localization of axonal proteins.</a> PLoS Biol. 9, (2010).</li><li>von Zastrow, M., Williams, 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=Modulating+neuromodulation+by+receptor+membrane+traffic+in+the+endocytic+pathway.">Modulating neuromodulation by receptor membrane traffic in the endocytic pathway.</a> Neuron. 76, 22-32 (2012).</li><li>Gunnersen, J. M., Augustine, C., Spirkoska, V., Kim, M., Brown, M., Tan, S. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+gene+expression+patterns+in+developing+mouse+neocortex+using+serial+analysis+of+gene+expression.">Global analysis of gene expression patterns in developing mouse neocortex using serial analysis of gene expression.</a> Mol. Cell. Neurosci. 19, 560-573 (2002).</li><li>Gunnersen, J. M., Kim, M. H., 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=Sez-6+proteins+affect+dendritic+arborization+patterns+and+excitability+of+cortical+pyramidal+neurons.">Sez-6 proteins affect dendritic arborization patterns and excitability of cortical pyramidal neurons.</a> Neuron. 56, 621-639 (2007).</li><li>Kennedy, M. J., Davison, I. G., Robinson, C. G., Ehlers, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syntaxin-4+Defines+a+Domain+for+Activity-Dependent+Exocytosis+in+Dendritic+Spines.">Syntaxin-4 Defines a Domain for Activity-Dependent Exocytosis in Dendritic Spines.</a> Cell. 141, 524-535 (2010).</li><li>Cullen, D. K., Gilroy, M. E., Irons, H. R., Laplaca, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapse-to-neuron+ratio+is+inversely+related+to+neuronal+density+in+mature+neuronal+cultures.">Synapse-to-neuron ratio is inversely related to neuronal density in mature neuronal cultures.</a> Brain Res. 1359, 44-55 (2010).</li><li>Grabrucker, A., Vaida, B., Bockmann, J., Boeckers, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptogenesis+of+hippocampal+neurons+in+primary+cell+culture.">Synaptogenesis of hippocampal neurons in primary cell culture.</a> Cell Tissue Res. , 338-341 (2009).</li><li>Vaillant, A. R., Zanassi, P., Walsh, G. S., Aumont, A., Alonso, A., Miller, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+mechanisms+underlying+reversible,+activity-dependent+dendrite+formation.">Signaling mechanisms underlying reversible, activity-dependent dendrite formation.</a> Neuron. 34, 985-998 (2002).</li><li>Park, M., Penick, E. C., Edwards, J. G., Kauer, J. A., Ehlers, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recycling+endosomes+supply+AMPA+receptors+for+LTP.">Recycling endosomes supply AMPA receptors for LTP.</a> Science. 305, 1972-1975 (2004).</li><li>Kennedy, M. J. &. a. m. p. ;., Ehlers, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelles+and+trafficking+machinery+for+postsynaptic+plasticity.">Organelles and trafficking machinery for postsynaptic plasticity.</a> Annu. Rev. Neurosci. 29, 325-362 (2006).</li><li>Lasiecka, Z. M., Yap, C. C., Caplan, S., Winckler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+early+endosomes+require+EHD1+for+L1/NgCAM+trafficking.">Neuronal early endosomes require EHD1 for L1/NgCAM trafficking.</a> J. Neurosci. 30, 16485-16497 (2010).</li><li>Arancibia-C&#225;rcamo, I. L., Fairfax, B. P., Moss, S. J., Kittler, J. T., Kittler, J. T., Moss, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+the+localization,+surface+stability+and+endocytosis+of+neurotransmitter+receptors+by+antibody+labeling+and+biotinylation+approaches.">Studying the localization, surface stability and endocytosis of neurotransmitter receptors by antibody labeling and biotinylation approaches.</a> The Dynamic Synapse: Molecular Methods in Ionotropic Receptor Biology. , (2006).</li><li>Petrini, E. M., Lu, J., Cognet, L., Lounis, B., Ehlers, M. D., Choquet, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytic+trafficking+and+recycling+maintain+a+pool+of+mobile+surface+AMPA+receptors+required+for+synaptic+potentiation).">Endocytic trafficking and recycling maintain a pool of mobile surface AMPA receptors required for synaptic potentiation).</a> Neuron. 63, 92-105 (2009).</li><li>Reider, A., Wendland, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytic+adaptors+&#8211;+social+networking+at+the+plasma+membrane.">Endocytic adaptors &#8211; social networking at the plasma membrane.</a> J. Cell Sci. 124, 1613-1622 (2011).</li></ol>

作者宣称，他们有没有竞争的财务权益。

这里所描述的技术是互补的细胞表面生物素化（由阿兰西维亚-CARCAMO 等人评论）12，它是所选择的用于保存关于内化蛋白的亚细胞定位信息的方法，提供一个合适的初级抗体，以一个胞表位是可用的。此外，蛋白质运输/内化的定量随着时间的推移可以执行（由整个活细胞温育与第一抗体固定盖玻片在不同时间），而不需要制备蛋白提取物。 染色强度或数量与泪...

在建立新发现的蛋白质的功能，有问题的蛋白质的亚细胞定位和贩运的调查可以提供有关蛋白质1,2的可能作用/ s的重要线索。显影皮层3的转录组的生物信息学分析为我们提供了基因在小鼠脑corticogenesis表现出改变的表达的列表。然后我们通过基因敲除的方法，以证实这些基因之一，sez6编码的蛋白质，具有在神经元发育的关键作用。我们观察到癫痫相关基因6，或Sez6，蛋白质位于开发树突和也存在于树突棘，在接收和集成的兴奋性信号的树突特殊结构。此外，当这种蛋白质缺乏，树突和兴奋性突触不能形成正确4。该蛋白质的可能优势亚型具有transmembran的特征Ë受体虽然，当免疫标记蛋白的亚细胞分布进行了检查，共聚焦显微镜或免疫电镜大多数，如果不是全部，信号的出现与在体树突车厢很少，或者没有，蛋白质标记在质膜上的小囊泡在细胞表面。 为了明确地表明，该推定受体与预测的大胞外结构域被贩卖到质膜上，我们使用我们已经生成的蛋白的胞外部分，以在细胞表面标记蛋白的抗血清，通过了活细胞的方法。通过组合这种“抗体馈送”的方法与差异标记的第二抗体的两个应用程序通过一个广泛的阻挡工序和透化步骤分开，我们能够确定蛋白质的区分两种不同的池通过结合到荧光标记的二级抗体轴承不同fluoresc耳鼻喉科标签。因此，我们能够区分在从蛋白质的抗体温育步骤，要么留在细胞表面上或在此期间被贩卖到表面已被内化由细胞内吞作用的蛋白质。使用这种方法，我们建立了所感兴趣的蛋白被贩卖到和从细胞表面的神经元。因此，这种相对快速和简单的技术证明比传统的免疫细胞化学方法或预嵌入免疫电镜更多的信息，尽管我们使用了相同的兔多克隆抗血清对所有这些技术的事实。该技术一般适用于任何跨膜蛋白提供了良好的抗体识别的细胞外结构域的表位是可用的。该技术已被以前用于研究谷氨酸受体的GluR1亚基5的受体转运。

<table border="0" cellpadding="0" cellspacing="0" width="937">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">PBS with Ca and Mg</td>
			<td style="width:269px;">Invitrogen</td>
			<td style="width:119px;">14040182</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Neurobasal medium</td>
			<td style="width:269px;">Invitrogen</td>
			<td style="width:119px;">21103-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">B27 supplement</td>
			<td style="width:269px;">Invitrogen</td>
			<td style="width:119px;">17504-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">L-glutamine</td>
			<td style="width:269px;">Invitrogen</td>
			<td style="width:119px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Papain Dissociation System</td>
			<td style="width:269px;">Worthington Biochemical Corporation</td>
			<td style="width:119px;">PDS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Bovine Serum Albumin&#160;</td>
			<td style="width:269px;">Sigma Aldrich Australia</td>
			<td style="width:119px;">A9418</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:383px;">Hank&#39;s balanced salt solution without calcium, magnesium, phenol red</td>
			<td style="width:269px;">Invitrogen (Gibco)</td>
			<td style="width:119px;">14175-079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Poly-D-Lysine</td>
			<td style="width:269px;">Sigma Aldrich Australia</td>
			<td style="width:119px;">P0899</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Natural mouse laminin</td>
			<td style="width:269px;">Invitrogen</td>
			<td style="width:119px;">23017-015</td>
			<td>Thaw on ice prior to making aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Fetal bovine serum HyClone</td>
			<td style="width:269px;">Thermo Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">fluorodeoxyuridine</td>
			<td style="width:269px;">Sigma Aldrich Australia</td>
			<td style="width:119px;">F0503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">uridine</td>
			<td style="width:269px;">Sigma Aldrich Australia</td>
			<td style="width:119px;">U3003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">18 mm round glass coverslips</td>
			<td style="width:269px;">Menzel Gl&#228;ser</td>
			<td>CB00180RA1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">VECTASHIELD aqueous mounting medium</td>
			<td style="width:269px;">Vector Laboratories</td>
			<td style="width:119px;">H1400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey anti-rabbit Dylight 649&#160;</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td style="width:119px;">711-495-152</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">AffiniPure Fab fragment Goat anti-Rabbit 1gG (H+L)&#160;</td>
			<td>Jackson ImmunoResearch Laboratories</td>
			<td>111-007-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Paraformaldehyde</td>
			<td style="width:269px;">Sigma Aldrich Australia</td>
			<td style="width:119px;">P6148</td>
			<td>TOXIC - handle in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:383px;">Triton-X-100</td>
			<td style="width:269px;">Sigma Aldrich Australia</td>
			<td style="width:119px;">T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexafluor 488-conjugated donkey anti-rabbit 2&#176; antibody</td>
			<td style="width:269px;">Invitrogen - Molecular Probes</td>
			<td style="width:119px;">A21206</td>
			<td></td>
		</tr>
	</tbody>
</table>

differential labeling of cell-surface and internalized proteins after antibody feeding of live cultured neurons

为了证明在培养的神经元的推定的跨膜受体的细胞表面定位，我们标记的蛋白活神经元的表面上与针对该蛋白的细胞外部分中的特定一抗。鉴于受体被贩卖到和从该表面，如果细胞被透化固定后那么这两个细胞表面和内部蛋白质将用相同的标记的第二抗体来检测。在这里，我们适于用来研究蛋白质运输（“抗体馈送”）进行差分时的抗体温育步骤和蛋白质，要么留在细胞表面上或在此期间被贩卖到表面已被内化通过内吞作用标签蛋白的方法。区分蛋白质的这两个池的能力成为可能通过阻断过夜与步高浓度未标记的二抗的初始incubatio后注册成立Ñ​​unpermeabilized神经元用荧光标记的第二抗体。经过封闭步骤，通透允许检测的内在池与标有不同的荧光团的荧光二抗神经元。使用这种技术，我们能够获得关于这一推定受体的亚细胞定位的重要信息，揭示它，的确，贩卖到细胞表面的神经元。这种技术广泛适用于各种细胞类型和细胞表面蛋白，从而提供一个合适的抗体的细胞外表位是可用的。

这里提出的双色荧光免疫染色技术是在活细胞标记的跨膜蛋白的细胞外结构域可使用（ 图1中示意性地示出）。在潜伏期，免疫球蛋白结合抗原表位访问和蛋白质分子的人口比例，与抗体结合在一起，被内吞。此外，新合成的蛋白质可通过向前贩卖到达细胞表面，并回收蛋白质分子可能返回至质膜10。 这种方法已被优化，两个不同的蛋白池，细胞表面的检...

Watch this Scientific Journal Video about Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons at JoVE.com

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

我们描述了一种方法，以使用特定的多克隆抗体对表位的细胞外活神经元的表面上的标记蛋白。蛋白结合的抗体在细胞表面上，并随后通过胞吞作用内在化可以从蛋白质上残留进行区分，或在培养过程中贩卖到，该表面。

细胞表面和内蛋白质的现场培养神经细胞的抗体后喂养差异化标记

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

differential-labeling-cell-surface-internalized-proteins-after

Research

JoVE Journal

Neuroscience

15.3K 次观看.  The University of Melbourne. 我们描述了一种方法，以使用特定的多克隆抗体对表位的细胞外活神经元的表面上的标记蛋白。蛋白结合的抗体在细胞表面上，并随后通过胞吞作用内在化可以从蛋白质上残留进行区分，或在培养过程中贩卖到，该表面。 

视频: Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

漂白后荧光标记蛋白的荧光恢复（FRAP）在培养海马神经元的树突棘

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

科研

神经科学

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

在培养的中枢神经系统的神经元树突棘形态分析

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

实时成像背根轴突后根切断术

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

诱导培养的交感神经元的树突状增长

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

从查明的神经元和取食行为的钙信号的同时记录果蝇</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

嵌入后突触蛋白的免疫标记海马切片培养

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

膜下丘脑腹内侧神经元的电位染料成像从成年小鼠为研究葡萄糖传感

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

使用荧光探针定量培养的神经元中的内体和溶酶体运动

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.