This work was supported by the NIDA Intramural Research Program (Bruce T. Hope, Yavin Shaham). F.J.R. was supported by an appointment to the NIDA Research Participation Program sponsored by the National Institutes of Health and administered by the Oak Ridge Institute for Science and Education, and received additional financial support from a Becas-Chile scholarship managed by CONICYT and the Universidad de los Andes, Santiago, Chile. The Johns Hopkins FACS Core facility was supported by Award P30AR053503 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institute of Health.

所有实验均按照健康指南全国学院的机构动物护理和使用委员会（IACUC）的护理和使用实验动物16进行。 注意:下面使用低结合离心管的所有步骤都保持在冰上，除非另有指定。 1.准备组织收集前<ol><li>离心机设置为4℃。 </li><li>火抛光具有大约1.3递减的直径，0.8和0.4毫米每个样品一组三个玻璃巴斯德移液管。 </li><li>准备含1毫升冷缓冲液A标1.7毫升管和保持管冰。 </li><li>制备含有脑切片矩阵，刮刀和其上以执行清扫玻璃板（或倒玻璃培养皿）的冰盘。预寒意两个或多个刀片上的玻璃板和使用组织纸晾干触及的T所有的仪器问题。 </li><li>解冻在室温（RT）处理30分钟组织收集前的酶溶液。 </li></ol> 2.组织收集和解剖<ol><li>发起组织收集前的行为和药物治疗90分钟。 注:对于这里示出的结果，5毫克/公斤的甲基苯丙胺对一种新的环境（测试条件）和幼稚大鼠大鼠急性腹膜内注射保持在它们的饲养笼（对照条件）进行。 </li><li>通过将其放置饱和异氟醚玻璃干燥器罐子麻醉大鼠和斩首大鼠30 - 60秒后用断头台。 用剪刀去除头部皮肤和肌肉，暴露颅骨。用咬骨钳切割颅骨和开拓枕骨大孔，取出头骨后面的部分。使用骨钳沿头骨的顶边切割以暴露脑。小心不要损伤大脑。使用小铲下轻轻舀Ð提高大脑。提高大脑和切断的神经，直到大脑是免费的17。 </li><li>使用刀片解剖组织。 注:获得使用以下两种方法的组织样本:（2.3.1）新鲜解剖组织; （2.3.2）解剖后冷冻的组织;或（2.3.3）组织冷冻冷冻全脑解剖。保持大脑切片矩阵，刀片和玻璃干板整个解剖和切碎流程作为的冷凝水可能会导致细胞的裂解低渗。使用放大镜，以确保准确的解剖。 <ol><li>对于新鲜解剖组织，将新鲜榨取的大脑已在冰上冷却大脑切片矩阵（大鼠脑形金属模具在1mm间隔的刀片插槽）。插入两个或更多个预冷刀片到槽切割包含所关注的脑区域（多个）冠状切片。放置在冷冻玻璃板切割片。 注:解剖大脑区域选择性对冷冻玻璃板兴趣纳秒。 </li><li>解剖后冷冻组织，从新鲜提取脑，类似于2.3.1上述切​​片解剖感兴趣的脑区域（多个）。放置解剖组织成微管并通过浸没在-40°异戊烷微管，持续20秒快速冷冻组织。保存在-80ºC冷冻，直到进一步的处理在任何时候都冻结解剖组织。 </li><li>对于冰冻组织，立即冻结在-40ºC异戊烷和储存在密封的袋子在-80ºC新鲜提取大脑长达6个月。 <ol><li>上解剖当天，将冷冻的脑在设定为约一低温恒温器-20℃（不超过华氏-18℃）约2小时，达到平衡的温度。 注:大脑可以很容易地在此温度下进行切割，而冷得多的温度使大脑太脆安全地切割。 </li><li>使用刀片切1 - 2毫米冠状切片冷冻大脑在低温恒温器。使用钝12至16号针头，以获得冷冻条件下，从这些切片组织拳。始终保持冷冻，直到进一步的处理解剖组织。 </li></ol></li></ol></li><li>添加1 - 缓冲液A 2滴切碎之前覆盖解剖组织。从使用两个刀片以防止磨碎处理的其余部分中的神经元损失的组织去除大部分的白质。如果与冷冻组织开始，然后允许组织向施加缓冲液A溶液之前在冷玻璃板解冻时间不超过1分钟。 </li><li>剁碎的组织在每个正交方向上的100倍以上的冷却玻璃板的剃刀刀片。切碎当握住刀片垂直于玻璃板。 注:彻底剁碎是所有协议的步骤至关重要。 </li><li>使用刀片的切碎的组织转移到含有1ml冷缓冲液A的倒置管3的离心管 - 5次使电机EP中的解决方案的所有组织糜。 </li></ol> 3.细胞分离注:使用温和的结束比上年末为以下所有步骤的混合。不要使用旋涡混合器，避免气泡造成的细胞损伤。 <ol><li>离心样品管在110 XG 2分钟，4ºC。弃上清。慢慢加入1毫升冷的刚解冻的酶溶液（含有蛋白水解酶的​​混合物）下微管的内壁。立即吸了整个颗粒和轻轻地用中等大小的尖端直径吸管吸取上下只有4次驱散组织糜颗粒。 </li><li>立即倒置微管，以防止粘附于底部的沉淀，并在4℃孵育与最终过端混合样品30分钟</li><li>酶组织消化后，离心机在4ºC960 XG 2分钟管子。除去上清液，加入0.6毫升冷的缓冲液A.立即出现的加入缓冲液A后Y，使用相同的枪头驱散颗粒通过吸了整个颗粒和吸管轻轻向上和向下的5倍。 </li><li>机械磨碎消化的组织，并在使用以下步骤15ml试管收集。对于每个样本，使用一套独立的3火抛光的玻璃巴斯德移液器降直径附着乳胶泡约1.3，0.8和0.4毫米。 <ol><li>轻轻磨碎每个样品与1.3毫米的玻璃吸移管的10倍。让样品静置2分钟在冰上（碎片和未离解的细胞将沉淀在底部）。收集上清液（〜0.6mL）中，并转移到15毫升锥形管（管＃1）。 </li><li>添加0.6毫升缓冲液中的溶液，以剩余的粒料。磨碎样品与0.8毫米的玻璃吸移管的10倍。让样品静置在冰上2分钟。收集上清液（〜0.6mL）中，并转移到同一的15毫升锥形管＃1。 </li><li>加入0.6毫升缓冲液的溶液将剩余PELLE吨。磨碎样品与0.4毫米的玻璃吸移管的10倍。让样品静置在冰上2分钟。收集上清液（〜0.6毫升;不接触与非解离的细胞沉淀），并转移到相同的15毫升锥形管＃1。 注意:保持0.4毫米直径的吸管在管＃1以下研磨步骤。 </li><li>重复步骤3.4.3三次用最小玻璃吸管（〜0.4mm直径），并且收集上清液到一个单独的15毫升锥形管（管＃2）。 </li><li>磨碎在管＃1和＃2所收集的细胞悬浮液，使用0.4毫米的玻璃吸移管10次以上，并保持在冰上。 </li></ol></li></ol> 4.细胞固定和透<ol><li>加入800微升100％的冷乙醇准备（从管＃2单元两个微管从管＃1单元和两个），每个样品4微管（储存在使用前-20℃）每管，并让他们在冰。注意:最终乙醇浓度为50％。 </li><li>吸取〜800微升细胞悬浮液（从管＃1和＃管2）到每个含冷乙醇的4管和颠倒管混匀样品。 </li><li>在冰上孵育管15分钟，同时反相，每5分钟的管中。 </li><li>固定/透化后，离心管1700 xg离心在4ºC4分钟并弃上清。离开50微升的溶液，以防止细胞的损失。 注:在此阶段的粒料是白色和粘比透以前少在管子的壁上。因此，小心地取出上清液通过触摸微管的管壁里没有颗粒和制定上清慢慢用微量。 </li></ol> 5.细胞过滤<ol><li>重悬来自步骤4.4用冷PBS将粒料如下: <ol><li>添加550微升冷PBS的两个微管，从管＃1来的一个，并使用适度大直径圆周佩特笔尖轻轻吸取细胞悬液上下5次。重复相同的一个微管由管＃2的到来。 </li><li>从管＃1细胞，使用相同的移液管，所述第一细胞悬浮液转移到第二沉淀。轻轻吹打组合的细胞悬液上下5次重悬第二沉淀。 </li><li>从管＃2细胞，重悬，并结合在5.1.2中描述的颗粒（ 即，第三和第四微管）。 </li></ol></li><li>过滤来自分别使用两种不同的对细胞滤器（100微米和40微米孔径大小）如下步骤5.1.2和5.1.3两种细胞悬浮液: <ol><li>预湿通过加入PBS至过滤器的内侧的每个细胞过滤。使用移液管从过滤器的外侧除去过量的PBS中。 </li><li>移液器步骤5.1.2细胞悬浮液均匀涂抹于100微米孔径的第一个细胞过滤。收集在冰上的50ml管中的流通。使用PI佩特收集液流 - 通过附着在细胞过滤的外侧底部。 </li><li>使用相同的移液管的流动通从100微米的细胞滤网转移到40微米的细胞滤网。收集在冰上另外50毫升管此流过。当40微米的细胞过滤通过传输流，切换到新的枪头，以避免从100微米的细胞过滤污染。 </li><li>重复这些步骤步骤5.1.3细胞悬液采用一套新的过滤器。 </li></ol></li><li>从每个样品结合两种流过为每个样品约1ml的最终体积。 </li></ol> 6.孵化与抗体注:使用脑细胞悬液的小等分，为运行主样本之前，适当的流式细胞仪的设置设置准备多个对照样品。对于每个抗体标记，准备对照样品，其中包括没有抗体，只有本身condary抗体（或其他荧光标记），然后一次和二次抗体（或其他荧光标签）。使用这些控件设置单独与特定的流式细胞仪的非特异性标记的大门。如果可能的话，使用不要求赔偿的荧光。加入第一抗体之前加入的PBS增加选通控制样品的700微升最终体积的最终体积。 <ol><li>通过过滤的细胞的100微升样品转移至1.7毫升微管制备光散射和DAPI对照样品。添加600微升冷PBS与1微克/毫升的DAPI对细胞核染色。孵育10分钟终过端的混​​合，并通过流过流式细胞仪之前清洗（如在步骤6.4.2至6.4.5表示）。 注:此示例只用于设置之前在FACS机器分拣剩余细胞的细胞/细胞核栅极。 </li><li>通过转让100＆＃制备单免疫标记对照样品181;微升过滤细胞对一个1.7毫升微型管。添加600微升冷PBS与一种抗体（ 例如，的NeuN抗体）。添加600微升冷PBS到与另一种抗体（ 例如，Fos抗体）微管。添加相应的二级抗体，如果它不是一直接缀合的初级抗体。 注意:这些样品被用来确定相应的光电倍增管（PMT）的电压，并在必要时，以评估两种荧光信号的重叠。抗体浓度应该滴定，以达到最佳的信号 - 噪声比中的所有信道。重叠的荧光信号，也可以通过流式细胞仪内不同的荧光通道的内部校准补偿。 </li><li>通过将100μl过滤的细胞转移到一个1.7毫升微型管制备双荧光标记的阴性对照样品。添加600微升冷PBS单独用二抗或IgG直接缀合的初级抗体。 注:氏与S样品可以进行排序，以确定每个荧光背景荧光之前使用每一次。 </li><li>制备主要样本进行排序。 <ol><li>转移700微升过滤细胞成1.7毫升微型管。加7微升（1:100）主Fos抗体的直接缀合的Alexa Fluor 647（抗的Fos-AF647）和1.4微升（1:500）初级的NeuN抗体的直接缀合到藻红蛋白（抗的NeuN-PE）。在4ºC与最终过端混合孵育管30分钟。 </li><li>在孵育结束时，加入800微升冷PBS到每个微管，包括主样品和对照样品，并通过倒置混合。 </li><li>微管离心在4ºC1300 XG 3分钟。弃去上清液，留下50微升上清液，以防止细胞的损失。 </li><li> 1毫升冷PBS添加到使用移液管有一个中等大小的顶端直径的颗粒和重悬细胞。 </li><li>离心机在4ºC1300 XG 3分钟。 ðiscard上清液。 </li><li>重悬在500微升冷PBS丸粒（调节终体积取决于颗粒的大小）。 </li><li>转移和悬浮液用细胞过滤网帽（40微米）过滤到圆底FACS样品管中。保持冰的免疫标记细胞和立即执行FACS。注意:对于过滤，垂直触及滤网盖枪头，并通过它推动细胞悬液。 </li></ol></li><li>设置流式细胞仪使用对照样品门。 <ol><li>使用光散射和DAPI控制样品从事件总数和细胞碎片识别神经元细胞群。 注:本制剂细胞体只有1 - 总的事件（其余的都是碎片和破碎的细胞过程）的2％。基于DAPI的荧光信号（指示核）和前向散射（小区大小），可以找到向后基于所述前向和侧向细胞体和栅极光散射特性将细胞（ 如图1的A，B）。 </li><li>使用单免疫标记对照样品，以确定PMT电压设定，并分析排放溢出从一个特定的荧光染料进入另一个过滤器/通道（ 如的Alexa Fluor 488 / FITC或藻红蛋白）。如果荧光信号重叠，通过手动调节薪酬水平纠正重叠。 </li><li>使用双荧光标记的阴性对照来建立阈值的正的人口。 注意:从该样品来的信号被认为是由于自体荧光和位于高于该阈值的主要试样的细胞可以被认为是阳性。通常情况下，用于排序的阈值的参数是更严格的，并大致标记的细胞的阴性对照以上的顶部2/3实际上排序。 </li></ol></li><li>排序由FACS主要样本的神经元到1.7毫升微管。收集细胞于50微升RNA的提取液。排序，涡旋并离心管并通过上下抽吸10次来恢复所有分选的细胞，从该管的壁混合该悬浮液后。 </li><li>孵育在42ºC的分选的细胞悬浮液30分钟，以确保所有的细胞之前，提取RNA裂解，然后在2,800 xg离心在4ºC离心2分钟。 </li><li>将上清转移到长期存储的无RNase的管中，在-80ºC或立即处理用于RNA分离，cDNA合成，靶基因的预扩增和qPCR样品，如先前13-15所述。 </li></ol>

<ol>
	<li>Bossert, J. M., 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=Ventral+medial+prefrontal+cortex+neuronal+ensembles+mediate+context-induced+relapse+to+heroin.">Ventral medial prefrontal cortex neuronal ensembles mediate context-induced relapse to heroin.</a> Nat Neurosci. 14, 420-422 (2011).</li><li>Cruz, F. C., 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=Role+of+nucleus+accumbens+shell+neuronal+ensembles+in+context-induced+reinstatement+of+cocaine-seeking.">Role of nucleus accumbens shell neuronal ensembles in context-induced reinstatement of cocaine-seeking.</a> J Neurosci. 34, 7437-7446 (2014).</li><li>Fanous, S., 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=Role+of+orbitofrontal+cortex+neuronal+ensembles+in+the+expression+of+incubation+of+heroin+craving.">Role of orbitofrontal cortex neuronal ensembles in the expression of incubation of heroin craving.</a> J Neurosci. 32, 11600-11609 (2012).</li><li>Koya, E., 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=Targeted+disruption+of+cocaine-activated+nucleus+accumbens+neurons+prevents+context-specific+sensitization.">Targeted disruption of cocaine-activated nucleus accumbens neurons prevents context-specific sensitization.</a> Nat Neurosci. 12, 1069-1073 (2009).</li><li>Cruz, F. C., Rubio, F. J., Hope, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+c-fos+to+study+neuronal+ensembles+in+corticostriatal+circuitry+of+addiction.">Using c-fos to study neuronal ensembles in corticostriatal circuitry of addiction.</a> Brain Research. 1628, 157-173 (2015).</li><li>Kamentsky, L. A., Melamed, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometric+cell+sorter.">Spectrophotometric cell sorter.</a> Science. 156, 1364-1365 (1967).</li><li>Kamentsky, L. A., Melamed, M. R., Derman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrophotometer:+new+instrument+for+ultrarapid+cell+analysis.">Spectrophotometer: new instrument for ultrarapid cell analysis.</a> Science. 150, 630-631 (1965).</li><li>Lobo, M. K., Karsten, S. L., Gray, M., Geschwind, D. H., Yang, X. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FACS-array+profiling+of+striatal+projection+neuron+subtypes+in+juvenile+and+adult+mouse+brains.">FACS-array profiling of striatal projection neuron subtypes in juvenile and adult mouse brains.</a> Nat Neurosci. 9, 443-452 (2006).</li><li>Lobo, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+profiling+of+striatonigral+and+striatopallidal+medium+spiny+neurons+past,+present,+and+future.">Molecular profiling of striatonigral and striatopallidal medium spiny neurons past, present, and future.</a> Int Rev Neurobiol. 89, 1-35 (2009).</li><li>Guez-Barber, D., 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=FACS+purification+of+immunolabeled+cell+types+from+adult+rat+brain.">FACS purification of immunolabeled cell types from adult rat brain.</a> J Neurosci Methods. 203, 10-18 (2012).</li><li>Guez-Barber, D., 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=FACS+identifies+unique+cocaine-induced+gene+regulation+in+selectively+activated+adult+striatal+neurons.">FACS identifies unique cocaine-induced gene regulation in selectively activated adult striatal neurons.</a> J Neurosci. 31, 4251-4259 (2011).</li><li>Fanous, S., 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=Unique+gene+alterations+are+induced+in+FACS-purified+Fos-positive+neurons+activated+during+cue-induced+relapse+to+heroin+seeking.">Unique gene alterations are induced in FACS-purified Fos-positive neurons activated during cue-induced relapse to heroin seeking.</a> J Neurochem. 124, 100-108 (2013).</li><li>Liu, Q. 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=Detection+of+molecular+alterations+in+methamphetamine-activated+Fos-expressing+neurons+from+a+single+rat+dorsal+striatum+using+fluorescence-activated+cell+sorting+(FACS).">Detection of molecular alterations in methamphetamine-activated Fos-expressing neurons from a single rat dorsal striatum using fluorescence-activated cell sorting (FACS).</a> J Neurochem. 128, 173-185 (2014).</li><li>Rubio, F. 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=Context-induced+reinstatement+of+methamphetamine+seeking+is+associated+with+unique+molecular+alterations+in+Fos-expressing+dorsolateral+striatum+neurons.">Context-induced reinstatement of methamphetamine seeking is associated with unique molecular alterations in Fos-expressing dorsolateral striatum neurons.</a> J Neurosci. 35, 5625-5639 (2015).</li><li>Li, X., 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=Incubation+of+methamphetamine+craving+is+associated+with+selective+increases+in+expression+of+Bdnf+and+trkb,+glutamate+receptors,+and+epigenetic+enzymes+in+cue-activated+fos-expressing+dorsal+striatal+neurons.">Incubation of methamphetamine craving is associated with selective increases in expression of Bdnf and trkb, glutamate receptors, and epigenetic enzymes in cue-activated fos-expressing dorsal striatal neurons.</a> J Neurosci. 35, 8232-8244 (2015).</li><li>US National Research Council. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the care and use of laboratory animals. , (2011).</li><li>Koya, E., Margetts-Smith, G., Hope, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Daun02+inactivation+of+behaviourally-activated+Fos-expressing+neuronal+ensembles.">Daun02 inactivation of behaviourally-activated Fos-expressing neuronal ensembles.</a> Current Protocols. , (2016).</li><li>Cruz, F. C., 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=New+technologies+for+examining+the+role+of+neuronal+ensembles+in+drug+addiction+and+fear.">New technologies for examining the role of neuronal ensembles in drug addiction and fear.</a> Nat Rev Neurosci. 14, 743-754 (2013).</li><li>Mardones, G., Gonzalez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+plasma+membrane+permeabilization+by+freeze-thawing+and+immunofluorescence+epitope+access+to+determine+the+topology+of+intracellular+membrane+proteins.">Selective plasma membrane permeabilization by freeze-thawing and immunofluorescence epitope access to determine the topology of intracellular membrane proteins.</a> J Immunol Methods. 275, 169-177 (2003).</li><li>Molyneaux, B. 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=DeCoN:+genome-wide+analysis+of+in+vivo+transcriptional+dynamics+during+pyramidal+neuron+fate+selection+in+neocortex.">DeCoN: genome-wide analysis of in vivo transcriptional dynamics during pyramidal neuron fate selection in neocortex.</a> Neuron. 85, 275-288 (2015).</li><li>Schwarz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+fluorescence+activated+cell+sorting+to+examine+cell-type-specific+gene+expression+in+rat+brain+tissue.">Using fluorescence activated cell sorting to examine cell-type-specific gene expression in rat brain tissue.</a> J Vis Exp. , e52537 (2015).</li></ol>

The authors declare that they have no competing financial interests.

FACS可用于从新鲜或冷冻的成人脑组织的神经元和其他细胞类型进行排序。如在引言中提到的，使用冷冻组织的能力允许从已经历复杂和长期行为的程序，如在成瘾研究自我施用和复发的研究的动物样品的最佳利用。这些行为过程通常需要1 - 2小时或更长的时间，并且要求所有动物（10 - 20个）上13,18在同一天进行测试。它需要〜4小时处理4新鲜解剖脑组织样本，其包括脑解剖，细胞解离，透化和免疫标记。处理后，FACS每个样本排序之间需要20 - 30分钟。在裂解缓冲液中加热分选的细胞的最后步骤需要30分钟。总共，〜7小时，从脑解剖需要最终含RNA的细胞裂解液的步骤。然而，现在有可能冻结整个brai纳秒或新鲜解剖大脑区域的测试后，立即和以后处理。这极大地简化了测试，并且允许多个脑区在不同的一天进行排序。冷冻前的FACS组织也可能会提高效果。更多的NeuN阳性神经元是从冷冻的组织样品获得。这可能部分地通过增加的NeuN标签由于冷冻和其后的解冻时细胞膜的破坏，这将增加抗体获得胞内蛋白如的NeuN和Fos蛋白进行说明。类似的效果已被观察到的成纤维细胞，上皮细胞和HeLa细胞19。 细胞和神经元的产率可以通过下列步骤被最大化。在组织剥离，（分别为胼胝体和前联合的背侧纹状体和伏隔核，）去除大部分的白质。这可以防止组织附着在随后STE塑料提​​示和玻璃吸管PS。使用缓冲液A（见材料和​​试剂表规范），以保持覆盖的解剖组织。这提高了具有刀片（步骤2.5）的组织的切碎过程中的细胞的质量。额外的一组具有最小玻璃吸管（步骤3.9）和随后的滤波带的第二组细胞滤器（步骤5）的3研磨步骤加倍细胞和神经元的产率。重用细胞过滤器会阻塞过滤器和较低的细胞产量。再悬浮颗粒和研磨期间，当温柔与细胞。在这些步骤中使用了中等大枪头将减少由于剪切应力细胞损伤。用乙醇（步骤4.4）透后，除去上清液时仔细观察沉淀。沉淀是不紧凑，并且可以沿着所述的离心管的壁分布。粗略的计算表明Fos蛋白阳性神经元的产率是解剖纹状体组织样品中的起始数的大约10％。 </p&gt; 目前的协议可以被修改以适应其它实验目的。在当前的协议，组织收集甲基苯丙胺注射之后90分钟，因为Fos表达达到在这个时候，它是Fos蛋白表达的神经元的有效排序的关键最大水平。然而，组织可以在用于FACS的任何时间点被收集，这取决于实验的具体目的。初始组织样品存储，我们已成功地使用新鲜解剖组织，解剖后冷冻组织，或从冷冻全脑解剖分离使用FACS随后的基因表达的定量PCR分析的Fos表达神经元冷冻的组织。在这个协议（4-ºC15分钟）固定和透化，持续时间和温度进行了优化，以透既细胞质和细胞核膜，以及以固定组织。在持续时间和/或温度变化可改变最终结果。其他固定的解决方案，如paraformaldehyde和非离子型去污剂如皂角苷，已对胚胎和产后神经元20的工作，也可能适用于从成年脑的神经元。此外，最近的研究显示用于FACS从脑组织21髓鞘去除珠可以用作当前协议的修改。 目前的协议有两个重要的考虑限制。首先，该协议不适合用于检测表达主要在突触（ 例如 ，多巴胺或谷氨酸受体），因为这些细胞过程是从在研磨和离解步骤胞体取出细胞表型标志物。第二个可能的限制是，从FACS分选的细胞中获得的RNA的长度可能不足以为所有RNA分析方法。用于从FACS获得的RNA的RNA完整性数（RIN）分选的细胞是2.5-3.5，其对应于较短的平均的RNA长度之间。在此，更短的RNA尺寸通过补偿使用80的相对较短的定量PCR扩增- 100个基点，如前面所描述13-15。我们也使用这些长度较短的RNA成功用于微阵列分析11。然而，获得的短RNA的长度可能不足以为所有RNA分析方法。应当强调的是，使用特异性靶向的外显子 - 外显子连接处仅在完全剪接的mRNA发现在胞质溶胶中的PCR引物。这些引物不从细胞核检测含内含子的RNA。此外，我们已经先前使用这个协议与抗体和新鲜的脑组织，以检测酪氨酸羟化酶在细胞质和D1多巴胺受体在细胞膜10。这些胞质和细胞膜标记物的检测表明，离解过程产生基本完好的细胞，而不是简单地细胞核。 总体上，从冷冻样品神经细胞的FACS隔离打开其他应用程序。在分拣或基因expre无差异裂变观察（数据未示出），当样品在-80储存3天或3周 ºC。三个星期是一个合理的时间从一个特定的实验中所有样本进行排序（20 - 40个样本）和基因表达分离RNA。可用的RNA也已经从在-80℃（数据未在这里示出）保存6个月的脑组织得到的。因此，该协议可能是已存储数月甚至数年验尸冷冻人脑样品的FACS隔离是有用的。

学习过程中，动物形成复台高度特异性的刺激之间的关联。此高分辨率信息被认为是由稀疏分布的神经元称为神经合奏特定图案内的改变进行编码。神经乐团最近已确定了的立即早期基因（即早基因）如Fos的 ， 圆弧和Zif268及其蛋白质产物在神经元的行为或线索暴露在强烈激活的诱导。 fos基因表达的神经元尤其已经示出在背景和线索特定习得行为1-4发挥因果作用。因此，这些被激活的Fos表达神经元内独特的分子neuroadaptations是编码了解到，在正常的学习和正常的学习障碍形成的协会，如成瘾和创伤后应激障碍（PTSD）5的神经机制的热门人选。 荧光李宗红激活细胞分选（FACS）最近允许的Fos蛋白表达的神经元内独特的分子neuroadaptations分析。流式细胞术和细胞分选在1960 6,7被开发根据它们的光散射和免疫特征来表征和分离细胞，并且早已在免疫学和癌症研究中使用。然而流式细胞仪和FACS需要解离的单细胞难以从成人脑组织得到。 FACS最早是用来分离和分析绿色荧光蛋白（GFP）-expressing从没有要求抗体标记8,9转基因小鼠纹状体神经元。我们开发了一种基于抗体的FACS方法10来隔离并在的Fos表达由药物和/或暗示在野生型动物中11-15激活的神经元评估分子改变。在该方法中，神经元被标以对一般的神经元标记物的NeuN的抗体，而强烈激活的神经元标记与抗Fos蛋白的抗体。虽然我们最初的方法所要求达到10只，每个样品新鲜组织池，该协议的后续修改允许从单个老鼠13-15的Fos表达神经元和定量聚合酶链反应（定量PCR）分立的大脑区域的分析，流式细胞仪分离。总体而言，在Fos蛋白表达的成瘾研究12,14,15各种上下文和提示激活行为的过程中激活的神经元中发现独特的分子变化。 与新鲜组织进行的FACS的一个主要的后勤问题是，它需要一整天以解离通过FACS组织和处理。此外，只有大约四个样品每天可处理。这通常意味着只有一个大脑区域可从各脑进行评估和剩余的脑区域都被丢弃。这对于低吞吐量行为程序，如自我施用和消光TRAI一个主要问题宁认为需要手术治疗和强化训练几个星期。此外，在测试当天长期和复杂的行为过程使得难以在同一天进行的FACS。这将是一个显著优点是能够从动物行为测试后，立即冻结的大脑，然后在研究者的选择的不同的时间从一个或多个脑区域隔离的Fos表达神经元。 在这里，我们证明，我们的FACS协议可以用于分离从新鲜和冷冻的脑组织的Fos表达神经元（和其它细胞类型）。作为一个例子，我们分离的Fos表达后急性注射甲基苯丙胺，并从幼稚大鼠不打针（对照条件）从纹状体神经元。然而，该FACS协议可以按照任何行为或药物治疗中使用。我们的样品的后续的qPCR分析表明，从这些类型的细胞的基因表达可以与西米进行评估从新鲜和冷冻组织征地拆迁的效率。

<table border="0" cellpadding="0" cellspacing="0" width="1274">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:455px;">Brain matrix</td>
			<td style="width:191px;">CellPoint Scientific</td>
			<td style="width:241px;">69-2160-1</td>
			<td style="width:388px;">to obtain coronal brain slices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hibernate A low fluorescence&#160;</td>
			<td>Brain Bits</td>
			<td>HA-lf&#160;</td>
			<td>Buffer A&#39; in the protocol is used for processing tissue and cells from the dissociation to fixation steps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Accutase&#160;</td>
			<td>Millipore</td>
			<td>SCR005</td>
			<td>Enzyme solution&#39; in the protocol is used for enzymatic digestion of tissue prior to trituration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein LoBind Tube 1.5 mL, PCR clean</td>
			<td>Eppendorf</td>
			<td>22431081</td>
			<td>to prevent cell lost during the protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Strainer, 40 &#181;m</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td>to filter cell suspension</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Strainer, 100 &#181;m</td>
			<td>BD Falcon</td>
			<td>352360</td>
			<td>to filter cell suspension</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon 5 ml round-bottom polystyrene test tube with cell strainer snap cap</td>
			<td>BD Bioscience</td>
			<td>352235</td>
			<td>to filter cell suspension before passing though the flow cytometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pasteur Pipet, Glass</td>
			<td>NIH supply</td>
			<td>6640-00-782-6008</td>
			<td>to do tissue trituration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Milli-Mark Anti-NeuN-PE, Clone A60 (mouse monoclonal) antibody</td>
			<td>EMD Millipore</td>
			<td>FCMAB317PE</td>
			<td>antibody to detect neurons</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phospho-c-Fos (Ser32) (D82C12) XP Rabbit (Alexa Fluor 647 Conjugate)&#160;</td>
			<td>Cell Signaling Technology&#160;</td>
			<td>8677</td>
			<td>antibody to detect Fos-expressing cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI</td>
			<td>Sigma</td>
			<td>D8417</td>
			<td>to label nuclei</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PicoPure RNA Isolation Kit</td>
			<td>Applied Biosystems.</td>
			<td>KIT0204</td>
			<td>The kit includes the RNA extraction buffer for step 6.14. It is used to collect sorted cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Superscript III first strand cDNA synthesis system</td>
			<td>Invitrogen</td>
			<td>18080-051</td>
			<td>to synthesize cDNA from RNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TaqMan PreAmp Master Mix</td>
			<td>Applied Biosystems.</td>
			<td>4391128</td>
			<td>to do targeted-preamplification from cDNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TaqMan Advance Fast Master&#160;</td>
			<td>Applied Biosystems.</td>
			<td>4444963</td>
			<td>to do PCR using TaqMan probes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fos TaqMan probe</td>
			<td>Applied Biosystems.</td>
			<td>Rn00487426_g1</td>
			<td>TaqMan probe/primers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NeuN TaqMan probe</td>
			<td>Applied Biosystems.</td>
			<td>CACTCCAACAGCGTGAC</td>
			<td>nucleotide sequence for neuronal gene marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NeuN Forward primer</td>
			<td>Applied Biosystems.</td>
			<td>GGCCCCTGGCAGAAAGTAG</td>
			<td>nucleotide sequence for neuronal gene marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NeuN Reverse primer</td>
			<td>Applied Biosystems.</td>
			<td>TTCCCCCTGGTCCTTCTGA</td>
			<td>nucleotide sequence for neuronal gene marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gfap TaqMan probe</td>
			<td>Applied Biosystems.</td>
			<td>Rn00566603_m1</td>
			<td>TaqMan probe/primers for astrocityc gene marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">iba-1 TaqMan probe</td>
			<td>Applied Biosystems.</td>
			<td>Rn00574125_g1</td>
			<td>TaqMan probe/primers for microglya gene marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gapdh TaqMan probe</td>
			<td>Applied Biosystems.</td>
			<td>CTCATGACCACAGTCCA</td>
			<td>nucleotide sequence for reference/housekeeping gene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gapdh Forward primer</td>
			<td>Applied Biosystems.</td>
			<td>GACAACTTTGGCATCGTGGAA</td>
			<td>nucleotide sequence for reference/housekeeping gene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gapdh Reverse primer</td>
			<td>Applied Biosystems.</td>
			<td>CACAGTCTTCTGAGTGGCAGTGA</td>
			<td>nucleotide sequence for reference/housekeeping gene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oligo2 TaqMan probe</td>
			<td>Applied Biosystems.</td>
			<td>Rn01767116_m1&#160;</td>
			<td>TaqMan probe/primers for oligodendrocytic gene marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P1000 pipettor</td>
			<td>Rainin</td>
			<td>17014382</td>
			<td>It is refered to as the pipette with a large tip diameter in steps 3.1 and 3.3 for mild tissue trituration and step 6.7 to resuspend cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7500 Fast Real-time PCR system</td>
			<td>Applied Biosystems.</td>
			<td>446985</td>
			<td>for quantitative PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACSAria I Cell Sorter</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>for FACS sorting</td>
		</tr>
	</tbody>
</table>

fluorescence activated cell sorting (facs) and gene expression analysis of fos-expressing neurons from fresh and frozen rat brain tissue

神经可塑性和习得行为的分子变化的研究是从全脑区的研究转向的稀疏分布激活的神经元称为介导学协会神经歌舞团特定集合的研究。荧光活化细胞分选（FACS）最近已经成年大鼠脑组织进行了优化，并允许使用抗神经元的NeuN标记和Fos蛋白，强烈地激活的神经元的标志物激活的神经元的隔离。到现在为止，Fos蛋白表达的神经元和其他细胞类型，从新鲜组织，从而享有很长的处理天脑样品的允许号码非常有限的冗长和复杂的行为过程之后进行评估隔离。在这里，我们发现，Fos蛋白表达来自背侧纹状体神经元和Fos蛋白的mRNA产率分别为新鲜解剖组织和组织在-80ºC冷冻3之间相似- 21天。另外，我们证实了表型所述的NeuN阳性和的NeuN阴性分选的细胞通过神经元的评估基因表达的（ 的NeuN），星形细胞（GFAP），少突胶质细胞（Oligo2）和microgial（Iba1）标记物，这表明冷冻组织也可用于的FACS分离的神经胶质细胞类型。总体而言，它是可以收集，解剖和冷冻多个FACS会话脑组织。这最大化从有价值动物受试者已经经常发生长和复杂的行为过程获得的数据量。

急性注射甲基苯丙胺后排序从单只新鲜和冷冻背侧纹状体组织的Fos阳性和Fos蛋白阴性神经元。 上述方案用于甲基苯丙胺（5毫克/千克）的腹膜内注射后从单个的大鼠背侧纹状体90分钟的Fos阳性和Fos蛋白阴性的神经元进行排序。在他们的家笼幼稚大鼠用作对照。背其集合（新鲜组织）或被冷冻并储存在-80ºC为1,7或21天之后进行处理后，立即进行处理纹状体组织任一。从这些冷冻时间点的结果不是彼此显著不同，所以该数据汇集。 要设置在仪器上的分选条件下，对​​照样品无论从家笼控制或methampheta（如上所述）用于矿山注入集团。当这些样品中的每个粒子或事件穿过所述流式细胞仪的细胞，它们被赋予与该事件的光散射和荧光特性相关联，并记录在电子表格中的识别号码。在此表中的事件可以然后在散点图或密度图，这些特性（ 图1）来组织。具有相似特征的事件可以被分组或由不同的成对的特性，以确定门如那些含有细胞，神经元，或Fos蛋白阳性神经元"门控"。一旦栅极使用上述的对照样品确定的，主样品按照这些门注入流式细胞仪和排序。细胞群体是从基于其正向光散射的所有事件（细胞和碎片）选通（FSC;粒子的大小的指示）和侧向光散射（SSC;粒子的克的指示ranularity）。如图中的FSC相对于SSC散点图（ 图1A和1B）中，所有事件的密度图揭示了具有相似尺寸和粒度小同质种群中，除了大的异质群体（ 图1A和1B）。包含细胞体小的同质群体被确定和门为"细胞"的基础上，FSC和SSC特征与以往研究11,13-15和DAPI标签。从比新鲜组织（1.9％），冷冻的组织（2.6％）获得细胞的比例略高。在大型异构人口主要是碎片，推测为树突和轴突的进程。 接着，从"细胞"门单个细胞进行鉴定根据它们的大小，这是通过对在x轴上的每个事件的FSC信号的宽度来表示。这比单细胞的大型活动被认为是细胞聚集<html>并排除在"单细胞门，所有的后续分析步骤，此范围内进行的"单个单元"门（ 图1C和1D）。此单细胞群的大部分（&gt; 92％）是阳性的DAPI染色（数据未示出）。 首先，神经元基于该指示的NeuN免疫反应（ 图1E和1F）他们的体育荧光强度确定。神经元从新鲜组织和冷冻组织38％的"单细胞"闸门包括所有事件的大约24％。几乎所有的在这个"神经元"门的事件（在新鲜组织为98％，在冷冻组织99％）呈阳性的细胞的细胞核（ 图1G和1H）的DNA的DAPI染色。在单细胞栅极剩余DAPI标记的事件的NeuN阴性和包括神经胶质细胞，少突胶质细胞和小胶质细胞，如通过qPCR在图3中证实。 ove_content"FO:保持-together.within页="1"&gt;然后神经元被列为的Fos阳性或Fos蛋白阴性的神经元基于它们的Alexa Fluor 647的荧光信号（Fos蛋白免疫反应）不同细胞类型的标记，Fos蛋白。在神经元的增加，由于不同的神经活动和时间过程的水平表达水平逐渐，因此，不会有对Fos蛋白阴性的神经元的Fos阳性之间的明确的阈值，在第一步骤（只用于离线分析，以计算的Fos阳性细胞）的百分比，我们定义）从幼稚家笼控制大鼠的NeuN阴性人口基于最大的Alexa-647荧光（对于Fos蛋白的Fos阳性神经元的阈值。Fos蛋白阳性神经元被表示在从图2中不同的实验条件下，蓝色正方形在新鲜和冷冻的组织中，从甲基苯丙胺注射的大鼠的百分比的Fos阳性神经元。（1.9 - 2.2％， 图2C和2D）是两次作为共mpared至家笼大鼠（0.7 - 0.8％， 图2A和2B）。 使用靶基因扩增前和RT-PCR的FACS分选的细胞，确认细胞类型特异性基因。 在第二步骤中，以确保从用于随后的mRNA主要样本仅Fos蛋白阳性神经元的排序分析和减少的Fos阴性细胞的夹杂物，Alexa的-647荧光阈值升高，使得Fos蛋白的仅上部三分之二在蓝色正方形阳性事件被分类收集。该阈值具有至少10倍大于上述用于计算样本中的Fos阳性神经元的百分比阈值更高的荧光（每个细胞更高Fos表达）。细胞的四群，从一个单一的组织样品，包括的NeuN阴性+ Fos蛋白阴性（〜5,000个事件），的NeuN阴性+的Fos阳性排序（介于25 - 83事件），的NeuN阳性+的Fos负ative（5000〜事件）和的NeuN阳性+ Fos蛋白阳性（44不等 - 为家庭组的133事件，185 - 450事件为甲基苯丙胺组）。第一，细胞类型特异性基因的NeuN阳性（包括的Fos阳性和Fos蛋白阴性）和的NeuN阴性群体（包括的Fos阳性和Fos蛋白阴性）的表达被证实（ 图3）。GAPDH为作为参照/看家基因的基础上，从我们以前的研究结果13。为了控制在PCR反应中的样品和cDNA在不同层次的RNA，使用两种靶基因和GAPDH cDNA的引物进行双链的qPCR反应。对于GAPDH看家基因的Ct值保持在15至31之间，用于精确测量。对新鲜和冷冻组织， 的NeuN mRNA水平在的NeuN阳性人群比的NeuN阴性群体（P &lt;0.05显著较大（约8倍）; 图3A </stroNG&gt;;数据来自家庭和甲基苯丙胺类相结合）。相反，对于新鲜和冷冻组织，GFAP（胶质细胞标记; 图3B）和Oligo2（少突胶质细胞标记物; 图3C）的mRNA水平比在的NeuN阳性人群（对更大的的NeuN阴性人口&lt;0.05 ）。在的NeuN阴性群体中观察到的mRNA水平，但这没有统计学显著;更高Iba1的类似的趋势（ 图3D小胶质细胞标记物）。载有无法在特定细胞类型来精确地测量特定mRNA的极低水平一些样品（ 例如 ，GFAP mRNA表达的NeuN标记的神经元）。最大的Ct值被定义为35，以排除过低被精确的检测值。较高的Ct值超过了我们的qPCR实验的置信度。 FOS mRNA表达从接收到的甲基苯丙胺的单次注射（ 图4）的大鼠中的NeuN阳性的神经元群检测。对于新鲜和冷冻组织，Fos的 mRNA水平比的Fos-负更大的神经元中的Fos阳性神经元（P &lt;0.05）。此外，虽然有在Fos蛋白阳性神经元从新鲜组织Fos蛋白 mRNA的3倍的增加，Fos蛋白 mRNA的Fos蛋白阳性神经元从冷冻的组织中检测到了11倍的增长。两者合计，这些mRNA的结果证实了Fos蛋白阳性神经元的身份，从新鲜和冷冻组织的Fos阴性的神经元和非神经元群体。此外，细胞类型特异性基因和感兴趣的其它基因也可从新鲜和冷冻的条件下，分选的细胞进行分析。 <img alt="图1" src="/files/ftp_upload/54358/54358fig1.jpg" /> 图1. 派息浇注从新鲜和冷冻鼠背侧纹状体sociated和标记的细胞。在&#39;细胞&#39;门被前向和侧向散射特性（AD）确定，由核DAPI染色（GH）阳性标记证实。在"细胞"人口中的"神经元"门是由荧光的NeuN为（; EF PE）来确定。 （A - B）的细胞门:所有事件的线性图，根据它们的前向散射（X轴，细胞大小）和侧向散射（Y轴，粒度）。 （C - D）单细胞闸门:AB中所示的电池门内前向散射高度（Y轴）和宽度（X轴）的线性图。 （E - F）的神经元栅极:免疫荧光对在光盘中所示的单电池内浇口PE-标记的NeuN（Y轴）的对数曲线图揭示了低级集群中的活动和非神经元细胞的上部集群中的神经元。 （G </stroNG&gt; - 高 ）核染色:对神经细胞内浇口DAPI标记的核（Y轴）荧光的对数曲线。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54358/54358fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54358/54358fig2.jpg" /> 图2. 基于双标签的的NeuN和Fos蛋白的新鲜和冷冻鼠背侧纹状体的Fos阳性和Fos蛋白阴性神经元的门控，从大鼠新鲜和冷冻背侧纹状体（单后直接从他们的家笼或90分钟取甲基苯丙胺的腹膜内注射）进行解离，并用抗的NeuN和Fos蛋白直接缀合的抗体标记，并使用FACS机器排序。排序是基于藻红蛋白（PE）标记的NeuN免疫荧光（X轴）和Alexa 647标记的Fos immunofluorescence（Y轴）。的Fos阳性（蓝点）和FOS阴性（红点）的神经元分别位于上和下右象限，分别。点图显示的NeuN阳性细胞（神经元）和的NeuN阴性细胞（灰色点）;蓝色的方块表示上述阈值Fos表达的神经元。 （A - B）家庭组:直接从他们的笼子采取天真老鼠。 （C - D）甲基苯丙胺类:接受甲基苯丙胺单一注射鼠。为FOS阳性神经元的阈值被选定为略高于最大的Alexa-647荧光（FOS-IR）的家笼对照组的NeuN阴性细胞中观察到。虽然0.7 - 所有神经元的0.8％的家庭组中的Fos阳性，1.9 - 所有神经元的2.2％的甲基苯丙胺类中的Fos阳性。 <img alt="图3" src="/files/ftp_upload/54358/54358fig3.jpg" /> 图3。 </在强&gt; 细胞类型特异性基因表达流式细胞分选，从新鲜和冷冻鼠背侧纹状体细胞。的NeuN阳性神经元（FOS阳性和Fos蛋白阴性）和的NeuN阴性细胞（FOS阳性和Fos蛋白阴性）进行了排序使用所描述的协议和细胞类型特异性基因的mRNA表达水平被用来确认细胞分选（A） 的NeuN为神经细胞的标记物。（B）的 GFAP是胶质细胞的标记物（C）Oligo2是标记为少突胶质细胞（D）Iba1为小神经胶质细胞的标记物。为的NeuN的mRNA，数据表示为平均值±SEM从新鲜组织相对表达水平倍值的NeuN阴性细胞（9 - 14）。对于GFAP（6 - 12），Oligo2（N = 9 - 11）和Iba1（N = 5 - 8）的mRNA，数据相对表现为平均倍数值的±SEM来表达水平在Neu从新鲜组织的N-阳性细胞。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54358/54358fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54358/54358fig4.jpg" /> 图4. 在苯丙胺注射。Fos蛋白阳性和Fos蛋白阴性的神经元之后，从新鲜或冷冻的大鼠背纹状体的FACS分选的神经元 的Fos mRNA水平如上述协议描述和Fos蛋白的mRNA水平证实了排序。数据以平均值±从新鲜组织相对表达水平折值的Fos阴性神经元（N = 3 - 4）SEM。

Watch this Scientific Journal Video about Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue at JoVE.com

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

在这里，我们提出了一个荧光活化细胞分选（FACS）协议来研究分子改建的Fos表达来自新鲜和冷冻的脑组织神经元歌舞团。使用冷冻组织可在多个会话许多脑区的FACS隔离，最大限度地利用宝贵的动物主题。

荧光活化细胞分选（FACS）和新鲜和冷冻大鼠脑组织的Fos表达神经元基因表达分析

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

fluorescence-activated-cell-sorting-facs-gene-expression-analysis-fos

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

29.1K 次观看.  NIDA, NIH, DHHS. 在这里，我们提出了一个荧光活化细胞分选（FACS）协议来研究分子改建的Fos表达来自新鲜和冷冻的脑组织神经元歌舞团。使用冷冻组织可在多个会话许多脑区的FACS隔离，最大限度地利用宝贵的动物主题。 

视频: Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

双荧光原位杂交食脑切片

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

科研

神经科学

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

优化小，解剖定义的大脑区域的DNA甲基化和基因表达分析

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the intestine and lower urinary tract. Both during development and in mature tissues these cells are often widely dispersed throughout tissues so that isolation of discrete populations using methods like laser capture micro-dissection is difficult. They can however be directly visualized by expression of fluorescent reporters driven from regulatory regions of neuron-specific genes like Tyrosine hydroxylase (TH). We describe a method optimized for high yields of viable TH+ neuronal progenitors from fetal mouse visceral tissues, including intestine and lower urogenital tract (LUT), based on dissociation and fluorescence-activated cell sorting (FACS). 
The Th gene encodes the rate-limiting enzyme for production of catecholamines. Enteric neuronal progenitors begin to express TH during their migration in the fetal intestine1 and TH is also present in a subset of adult pelvic ganglia neurons2-4 . The first appearance of this lineage and the distribution of these neurons in other aspects of the LUT, and their isolation has not been described. Neuronal progenitors expressing TH can be readily visualized by expression of EGFP in mice carrying the transgene construct Tg(Th-EGFP)DJ76Gsat/Mmnc1. We imaged expression of this transgene in fetal mice to document the distribution of TH+ cells in the developing LUT at 15.5 days post coitus (dpc), designating the morning of plug detection as 0.5 dpc, and observed that a subset of neuronal progenitors in the coalescing pelvic ganglia express EGFP. 
To isolate LUT TH+ neuronal progenitors, we optimized methods that were initially used to purify neural crest stem cells from fetal mouse intestine2-6. Prior efforts to isolate NC-derived populations relied upon digestion with a cocktail of collagenase and trypsin to obtain cell suspensions for flow cytometry. In our hands these methods produced cell suspensions from the LUT with relatively low viability. Given the already low incidence of neuronal progenitors in fetal LUT tissues, we set out to optimize dissociation methods such that cell survival in the final dissociates would be increased. We determined that gentle dissociation in Accumax (Innovative Cell Technologies, Inc), manual filtering, and flow sorting at low pressures allowed us to achieve consistently greater survival (&gt;70% of total cells) with subsequent yields of neuronal progenitors sufficient for downstream analysis. The method we describe can be broadly applied to isolate a variety of neuronal populations from either fetal or adult murine tissues.

An Optimized Procedure for Fluorescence-activated Cell Sorting FACS Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice

An optimized procedure to purify neural crest-derived neuronal progenitors from fetal mouse tissues is described. This method takes advantage of expression from fluorescent reporter alleles to isolate discrete populations by fluorescence-activated cell sorting (FACS). The technique can be applied to isolate neuronal subpopulations throughout development or from adult tissues.

荧光激活细胞分选（FACS）自主神经祖细胞的分离，从胎鼠的内脏器官的优化程序

During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

激光捕获显微切割的丰富种群的创伤性脑损伤后神经元或单个神经元的基因表达分析

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

对基因差异表达的RNA序列中电穿孔鸡胚脊髓分析

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

基因表达的变化大鼠海马后，前丘脑核脑深部刺激分析

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

在大鼠脑组织用荧光活化细胞分选，以检查细胞类型特异性基因表达

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

新鲜人脑组织中毛细血管的分离

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

激光捕获显微切割分离新鲜冷冻人体肠神经节的优化

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can be cultured in defined media for 16 days in vitro and are amenable to any analyses typically performed on dissociated cultures, including electrophysiological, morphological and survival analyses. The major advantage of these cultures is that specific cell types can be selectively studied in the absence of complex external influences, such as those arising from glial cells or other neuron types. When planning experiments with purified cells, however, it is important to note that neurons strongly depend on glia-conditioned media for their growth and survival. In addition, glutamatergic neurons further depend on glia-secreted factors for the establishment of synaptic transmission. We therefore also describe a method for co-culturing neurons and glial cells in a non-contact arrangement. Using these methods, we have identified major differences between the development of GABAergic and glutamatergic neuronal networks. Thus, studying cultures of purified neurons has great potential for furthering our understanding of how the nervous system develops and functions. Moreover, purified cultures may be useful for investigating the direct action of pharmacological agents, growth factors or for exploring the consequences of genetic manipulations on specific cell types. As more and more transgenic animals become available, labeling additional specific cell types of interest, we expect that the protocols described here will grow in their applicability and potential.

This protocol describes a cell sorting based method for the purification and culture of fluorescent GABAergic or glutamatergic neurons from the neocortex and hippocampus of postnatal mice or rats.

荧光活化细胞分选建立纯化的 Gabaep 或谷胱甘肽神经元的原代细胞培养

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can ...