The authors thank S. Tausch for excellent technical assistance. The work was supported by BMBF (Bernstein Focus 01GQ0923) and DFG (FOR1738).

注：所有涉及活体动物程序进行了按照欧盟指令的护理和使用实验动物的六百○九分之八十六/ EEC的准则，并经当地伦理委员会（图林根Landesamt献给Lebensmittelsicherheit UND Verbraucherschutz）。 胸苷类似物1.腹腔注射<ol><li>注射前称量动物的一天。计算的胸苷类似物所需计划第二天以及10毫克/毫升储液个体体重调整注射量所有注射的量。 </li><li>制备10毫克/毫升胸苷原液。 （ 注意！胸苷类似物是有毒的。按照供应商所提供的具体材料安全数据表（MSDS）， 即穿上白大褂和手套，使用化学通风柜）。 <ol><li>就拿胸苷模拟从冰箱中，并把它带到RT，约21°C。体重10毫克，并添加无菌生理盐水（对于尿嘧啶和CldU）或0.04的1N NaOH（在无菌盐水;对于IDU），旋涡。放置至少10 - 15分钟，在50℃水浴和涡每2 - 3分钟，以溶解粉末。 当存储在室温和几个星期在-20°C使用的解决方案长达24小时：注意。从灯（盖有铝箔）保护的解决方案。务必检查沉淀物，如果需要重新溶解。 </li></ol></li><li>由浮渣抑制小鼠和腹腔内注入用细剂量注射器与地下30针原液（在RT）的一个适当的，体重调整音量。 注意：在壳体CldU和IDU必须顺序地在同一动物体内给药时，考虑到注入等摩尔浓度（ 例如，42.5毫克/千克CldU和57.5毫克/公斤IDU，其对应于50毫克/公斤的BrdU）。因此，相应地调整的10毫克/毫升储液的注射量。 </li></ol> 2.组织准备<ol><li>准备相再在0.1M磷酸盐缓冲液pH 7.4的4％甲醛在灌注前一天。保存在4℃。 </li><li> Transcardially灌注通过左心室的深度麻醉小鼠（3.5％异氟烷），用10ml冰冷的PBS，然后用40毫升冰冷的甲醛（流速为5毫升/分钟）。解剖脑和定位后，在同一固定液为24小时，在4℃下。 </li><li>大脑转移连续成10％（24小时，在4℃下）和30％的蔗糖（直到大脑水槽，约48小时）。对于冻结，慢慢淹没冷冻保护大脑到-25°C至异戊烷没有气泡从组织出现。储存在-80℃。 </li><li>切为40μm厚的冷冻切片机冠状切片（块温度在-25至-16℃）。依次转印部到含24孔细胞培养板的孔中的防冻溶液（参见图1）。存储在-20℃。 </li></ol><img aLT =“图1”SRC =“/文件/ ftp_upload / 52551 / 52551fig1.jpg”/&gt; 转移切片切片放入24孔板图1.示意图。开始在A1，并将随后的片，放入A行，之后A6到下一行B等等。当达到D6，回去A1和继续。切片的这种布置允许每n 个一个整个大脑的部分的定量。新生细胞的定量取每6 个脑切片（相当于一列的内容），进行免疫荧光表型取每12 个部分（相当于第2交替行的一列的内容）。 3.免疫染色注：第被处理自由浮动，通常在6孔板配有载体板和网状插入物。作为例外，阻塞，抗体孵育和ABC的反应是在12或24孔板无网格插入（每瓦特0.5至1ml做ELL是足够的，这取决于要被着色）的片数。在这些步骤中，转移部分用细刷子的帮助下（冲洗每一个新的解决方案）。所有温育完成连续搅拌（最大150rpm下）。 <ol><li> 免疫组化（ABC法） <ol><li>从转移防冻液部分进入TBS进行彻底的冲洗（一旦O / N在4°C，5次RT，每次10分钟），以彻底清除防冻液。 </li><li>孵育在1.5％的H 2 O 2的30分钟在TBS-T中以猝灭内源性过氧化物酶的活性。注意起泡，如有必要重新淹没部分。冲洗在TBS中3次，每次15分钟。 </li><li> 可选：同时预热加热箱和2 ​​N盐酸至37℃。孵育切片30分钟，在37℃，在2当量盐酸，以使DNA变性。轻轻的独立部分，用刷子的帮助。 </li><li> 可选：在0.1M硼酸盐缓冲液中和节10分钟8.5，RT。在传输，简单地擦拭含在纸巾上的部分，以除去HCl残渣网状嵌件。漂洗2次的TBS为每15分钟。 </li><li>孵育在TBSplus通透组织并阻断非特异性抗体结合位点，1小时，在室温。 </li><li>孵育在4℃稀释TBSplus，O / N一抗。冲洗在TBS中3次，每次15分钟。 </li><li>孵育稀释TBSplus，3小时，在RT生物素化的第二抗体。冲洗在TBS中3次，每次15分钟。同时... </li><li>根据制造商的协议（在TBS-T中的1％A + 1％B）的制备ABC复合物。允许在使用前在RT下放置30分钟。孵育AB试剂节1小时在室温。冲洗在TBS中3次，每次15分钟。 </li><li>制备50毫升0.5毫克/毫升DAB在TBS-T中每6-或12孔板中，分为两半，并吸移管4毫升或2毫升每孔分别。转移部分到DAB溶液（ 注意！DAB是有毒的。按照specific。通过供应商提供的， 也就是说，穿上白大褂和手套MSDS，使用通风橱）。 </li><li>加入0.5毫升1％的H 2 O 2的剩余的25毫升DAB溶液，混合和吸管等体积如上到每个孔中以开始过氧化物酶反应。孵育12分钟。冲洗在TBS中3次，每次15分钟。 </li><li>安装部分，以幻灯片的明胶，空气干燥O / N。盖玻片永久安装中。 </li><li> 可选：将盖玻片前染液（参见3.5节）。 注意：如果信噪比是由于高背景低，具有AB试剂（步骤3.1.8）孵育后重复的 H 2 O 2的处理。 </li></ol></li><li> 多重免疫 <ol><li> 单个或同时进行多个免疫 <ol><li>从转移防冻液部分进入TBS进行彻底的冲洗（一旦O / N在4°C，5次在室温10分钟每次）彻底删除tifreeze。 </li><li> 可选：作为步骤3.1.3 - 3.1.4。 </li><li>孵育在TBSplus通透组织并封闭非特异性抗体结合位点，1小时，在室温。 </li><li>在孵育初级抗体鸡尾酒（ 例如，大鼠α-尿嘧啶，豚鼠α-DCX，山羊α-GFP）在4℃稀释TBSplus，O / N。冲洗在TBS中3次，每次15分钟。 </li><li>在孵育荧光标记的二次抗体鸡尾酒（ 例如，罗丹明红α-鼠，Alexa的-647α-豚鼠，Alexa的-488α-山羊;所有派生的驴）稀释TBSplus，3小时在室温或O / N在4℃下。从现在开始保护部分由轻。冲洗在TBS中3次，每次15分钟。 </li><li>安装部分，以幻灯片的明胶，空气干燥O / N。盖玻片与水性封固剂。 </li></ol></li><li> 连续多次免疫与来自同一主机物种的主要抗体 <ol><li>作为步骤3.2.1.1 - 3.2.1.3。 </li><li>孵育科幻第一个主要的抗体（ 如兔α-抗原A），O / N在4℃。冲洗在TBS，一次3次在TBS-T中，每次10分钟。 </li><li>孵育在第一荧光标记的二抗（ 例如，若丹明红连接词。驴α-兔），3小时室温。从现在开始保护部分由轻。冲洗在TBS，一次3次在TBS-T中，每次10分钟。 </li><li>孵育在从3小时的RT同一主机作为主要的抗体（ 例如，兔血清）的10％正常血清饱和开放互补位上的第一次级抗体。冲洗在TBS，一次3次在TBS-T中，每次10分钟。 </li><li>孵育在TBSplus用50微克针对该初级抗体的宿主（ 例如，α -兔IgG（H + L）），以支付表位，可以通过对第二个二次抗体，O / N在被识别/ ml的未偶联的单价Fab片段4℃。 </li><li>在TBS-T中，每次10分钟，冲洗在TBS和一次至少3次。而转移，简要SWAB含有在纸巾的部分网状刀片，以除去任何的Fab残渣。 </li><li>孵育在第二初级抗体（ 如兔α-抗原B），O / N在4℃。冲洗在TBS，一次3次在TBS-T中，每次10分钟。 </li><li>孵育在第二荧光标记的第二抗体（ 例如，的Alexa-488-连接词。驴α-兔），3小时室温。在冲洗TBS 3次，每次15分钟，并安装如上面盖玻片。 注意：要标记抗原的抗体来自不同宿主物种将它们添加到步骤3.2.2.2和各二级抗体步骤3.2.2.3。 </li></ol></li><li> 一种从相同物种二次抗体作为初级抗体之一 注：此协议适用于对尿嘧啶或Ki67的四倍染色与GFAP，巢蛋白GFP和DCX。 <ol><li>严格遵循协议的步骤3.2.1.1 - 3.2.1.5。直到此时在TBSplus只使用驴血清。 </li><li>孵育T中BSplus含3％山羊血清1小时，在室温。这包括对α-山羊二次抗体开互补位。冲洗在TBS，一次3次在TBS-T中，每次10分钟。 </li><li>孵育AMCA-连词。山羊α兔稀释TBSplus，3小时RT或O / N 4℃。冲洗在TBS中3次，每次15分钟，并装入如上述盖玻片。 </li></ol></li><li> CldU，IDU共染色 <ol><li>漂洗，变性和在3.2.1节中描述中和节（步骤3.2.1.1 - 3.2.1.2）。 </li><li>孵育20微克/毫升未缀合的Fabα - 小鼠IgG（H + L）在TBSplus，1小时，在室温片段。冲洗4次的TBS，一次在TBS-T中，每次10分钟。 </li><li>孵育在含有鼠α-BrdU的初级抗体混合物（1：400;纯化的IgG2）和小鼠α-的BrdU（1：350），在4℃稀释在TBSplus，O / N。冲洗在TBS，一次3次在TBS-T中，每次10分钟。 </li><li>在孵育二级抗体鸡尾酒含生物素化的驴α-大鼠（1：500）和FITC-连接词。驴α - 小鼠Fab片段（1：100）。冲洗在TBS，一次3次在TBS-T中，每次10分钟。 </li><li>在孵育罗丹明红连词。链亲和素，2小时，在RT。在冲洗TBS 3次，每次15分钟，并安装如上面盖玻片。 </li></ol></li><li> 荧光信号的巢蛋白-GFP小鼠扩增 <ol><li>加入山羊α-GFP与一级抗体的鸡尾酒。 </li><li>添加荧光标记的第二抗体具有相似于GFP的光谱性质（例如，页面488连接词。驴α-山羊）到二次抗体混合物。 </li></ol></li></ol></li><li> 表位检索 <ol><li>冲洗出来后，防冻液进行表位检索。预热蒸锅用含有0.1M柠檬酸缓冲液pH为6.0至95 6或12孔板 - 99℃（约25分钟）。 </li><li>转移部分成热柠檬酸盐缓冲液和蒸汽进行30分钟（用铝箔覆盖板的塑料升IDS是不耐热）。 </li><li>立即将板在冰浴中冷却下来。这有助于保护组织的形态，与产后动物的脑切片的工作时，这是特别重要的。 </li><li>冲洗在TBS中3次，每次10分钟，以冲洗和中和柠檬酸，继续染色。 </li></ol></li><li> 鼠抗体的小鼠组织 <ol><li>加20微克/毫升的单价Fab片段α -小鼠IgG（H + L;同一宿主物种比二次抗体）与第一阻挡步骤（ 例如，步骤3.1.5或3.2.1.3）。 </li><li>冲洗4次的TBS，一次在TBS-T中，每次10分钟，并继续与各个协议。 </li></ol></li><li> 甲酚紫复染 <ol><li>预热甲酚紫溶液至60℃（在玻璃瓶中）。在3分钟的热溶液中孵育载玻片。 </li><li>在冲洗水溶液。 dest中。和脱水2次，每次1分钟，在70％，96％和100％异丙醇。 </l我&gt; <li>清除5 - 6分钟二甲苯或二甲苯替代和盖玻片与兼容的永久安装中。 </li></ol></li></ol> 4.数据分析<ol><li>计数沿齿状回的整个rostrocaudal程度每6 个部分中的过氧化物酶染色的新生细胞。使用光学显微镜的放大400倍。 </li><li>乘所得的细胞数与相交间隔，以获得总的数字新生细胞的估计。 </li><li>图像中的荧光标记的部分用激光共聚焦显微镜配备适当的激光器和过滤系统。取图像堆栈在沿齿状回在400X放大率的整个程度的随机位置，并分析每个半球兴趣用于共标记与其它标记物的至少50个随机选择的细胞。 </li><li>乘所得的共标记的细胞（％/ 100）的百分比与总数新生细胞来计算绝对具体新生细胞群的编号。 </li></ol>

<ol>
	<li>Kempermann, G., Jessberger, S., Steiner, B., Kronenberg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milestones+of+neuronal+development+in+the+adult+hippocampus.">Milestones of neuronal development in the adult hippocampus.</a> Trends Neurosci. 27 (8), 447-452 (2004).</li><li>Ge, S., Sailor, K. A., Ming, G. L., Song, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+integration+and+plasticity+of+new+neurons+in+the+adult+hippocampus.">Synaptic integration and plasticity of new neurons in the adult hippocampus.</a> J Physiol. 586 (16), 3759-3765 (2008).</li><li>Ge, S., Yang, C. H., Hsu, K. S., Ming, G. L., Song, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+period+for+enhanced+synaptic+plasticity+in+newly+generated+neurons+of+the+adult+brain.">A critical period for enhanced synaptic plasticity in newly generated neurons of the adult brain.</a> Neuron. 54 (4), 559-566 (2007).</li><li>Castilla-Ortega, E., Pedraza, C., Estivill-Torrus, G., Santin, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=When+is+adult+hippocampal+neurogenesis+necessary+for+learning?+evidence+from+animal+research.">When is adult hippocampal neurogenesis necessary for learning? evidence from animal research.</a> Rev Neurosci. 22 (3), 267-283 (2011).</li><li>Deng, W., Aimone, J. B., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+neurons+and+new+memories:+how+does+adult+hippocampal+neurogenesis+affect+learning+and+memory.">New neurons and new memories: how does adult hippocampal neurogenesis affect learning and memory.</a> Nat Rev Neurosci. 11 (5), 339-350 (2010).</li><li>Encinas, J. M., Enikolopov, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+and+quantitating+neural+stem+and+progenitor+cells+in+the+adult+brain.">Identifying and quantitating neural stem and progenitor cells in the adult brain.</a> Methods Cell Biol. 85, 243-272 (2008).</li><li>Burns, K. A., Kuan, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+doses+of+bromo-+and+iododeoxyuridine+produce+near-saturation+labeling+of+adult+proliferative+populations+in+the+dentate+gyrus.">Low doses of bromo- and iododeoxyuridine produce near-saturation labeling of adult proliferative populations in the dentate gyrus.</a> Eur J Neurosci. 21 (3), 803-807 (2005).</li><li>Cameron, H. A., McKay, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neurogenesis+produces+a+large+pool+of+new+granule+cells+in+the+dentate+gyrus.">Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus.</a> J Comp Neurol. 435 (4), 406-417 (2001).</li><li>Vega, C. J., Peterson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+proliferative+history+in+tissue+revealed+by+temporal+halogenated+thymidine+analog+discrimination.">Stem cell proliferative history in tissue revealed by temporal halogenated thymidine analog discrimination.</a> Nat Methods. 2 (3), 167-169 (2005).</li><li>Scholzen, T., Gerdes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ki-67+protein:+from+the+known+and+the+unknown.">The Ki-67 protein: from the known and the unknown.</a> J Cell Physiol. 182 (3), 311-322 (2000).</li><li>Brown, D. C., Gatter, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ki67+protein:+the+immaculate+deception.">Ki67 protein: the immaculate deception.</a> Histopathology. 40 (1), 2-11 (2002).</li><li>Yamaguchi, M., Saito, H., Suzuki, M., Mori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+neurogenesis+in+the+central+nervous+system+using+nestin+promoter-GFP+transgenic+mice.">Visualization of neurogenesis in the central nervous system using nestin promoter-GFP transgenic mice.</a> Neuroreport. 11 (9), 1991-1996 (2000).</li><li>Kempermann, G., Kuhn, H. G., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+hippocampal+neurons+in+adult+mice+living+in+an+enriched+environment.">More hippocampal neurons in adult mice living in an enriched environment.</a> Nature. 386 (6624), 493-495 (1997).</li><li>Sicinski, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin+D2+is+an+FSH-responsive+gene+involved+in+gonadal+cell+proliferation+and+oncogenesis.">Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis.</a> Nature. 384 (6608), 470-474 (1996).</li><li>Ansorg, A., Witte, O. W., Urbach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+kinetics+of+dentate+gyrus+neurogenesis+in+the+absence+of+cyclin+D2.">Age-dependent kinetics of dentate gyrus neurogenesis in the absence of cyclin D2.</a> BMC Neurosci. 13, 46 (2012).</li><li>Taupin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BrdU+immunohistochemistry+for+studying+adult+neurogenesis:+paradigms,+pitfalls,+limitations,+and+validation.">BrdU immunohistochemistry for studying adult neurogenesis: paradigms, pitfalls, limitations, and validation.</a> Brain Res Rev. 53 (1), 198-214 (2007).</li><li>Lewis Carl, S. A., Gillete-Ferguson, I., Ferguson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+indirect+immunofluorescence+procedure+for+staining+the+same+cryosection+with+two+mouse+monoclonal+primary+antibodies.">An indirect immunofluorescence procedure for staining the same cryosection with two mouse monoclonal primary antibodies.</a> J Histochem Cytochem. 41 (8), 1273-1278 (1993).</li><li>Burry, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controls+for+immunocytochemistry:+an+update.">Controls for immunocytochemistry: an update.</a> J Histochem Cytochem. 59 (1), 6-12 (2011).</li><li>Tuttle, A. 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=Immunofluorescent+detection+of+two+thymidine+analogues+(CldU+and+IdU)+in+primary+tissue.">Immunofluorescent detection of two thymidine analogues (CldU and IdU) in primary tissue.</a> J Vis Exp. (46), 6-12 (2010).</li><li>Miller, R. T., Swanson, P. E., Wick, 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=Fixation+and+epitope+retrieval+in+diagnostic+immunohistochemistry:+a+concise+review+with+practical+considerations.">Fixation and epitope retrieval in diagnostic immunohistochemistry: a concise review with practical considerations.</a> Appl Immunohistochem. Mol Morphol. 8 (3), 228-235 (2000).</li></ol>

The authors declare that they have no competing financial interests.

量化和鉴定新生细胞亚群是在成年神经研究的一个核心问题。结合增殖标记和抗体在成年神经的特定阶段表达的蛋白质可以让免疫组化检测这些亚群。一些抗体或抗体组合的需要特定的染色条件。 分裂细胞合成胸腺嘧啶核苷类似物的标记仍然是金标准研究成年海马神经发生。关键的是要考虑到开始实验前适当注射协议。我们通常施用50毫克/千克（体重，IP）的BrdU，但浓度不?...

两个脑区的组成产生整个人生新的神经元，侧脑室的脑室下区和海马齿状回（DG）的颗粒下区（SGZ）。新生神经元的神经祖细胞获得茁壮1,2之前通过形态和生理发育的不同阶段。从缓慢除以径向胶质样干细胞（1型）的转运连续阶段扩增中间祖细胞产生。更未分化的亚型（式2a和式2b）中具有不规则的形状，总之，切向流程。它们所产生的神经母细胞（类型3），该逐步退出细胞周期成为未成熟神经元（与树突伸向分子层），最后整合进海马网络作为成熟粒细胞。由于其特殊的生理特点，这些细胞提供电路具有增强的可塑性3 sugges婷在海马功能独特的作用。事实上，在过去十年的研究产生的大量证据表明，成年神经有助于空间记忆，模式分离和情感行为4,5。 成年神经可以用不同的方法进行研究。胸苷类似物掺入到DNA中的S期的细胞周期，并允许新生细胞6-8出生约会，定量和命运分析。不同胸苷类似物（ 例如，CldU，埃杜或IDU）的顺序应用可以用于研究细胞周转或实验9的过程中，出生在不同时间点的细胞群。另一种，内源性标志物细胞增殖Ki67的。它表达于在细胞周期（G1，S，G2，M），除休止期（G0）及G1 10,11的开始的各个阶段的分裂细胞。分析新生细胞群的表型的成人dentat在Ë回几个阶段特异性的分子标记可用于如GFAP，巢蛋白，DCX和的NeuN 1,6。 GFAP是星形胶质细胞成熟的标志，但也表达了放射状胶质细胞样细胞在成人脑。巢蛋白是特异的放射状胶质细胞样细胞和早期中间祖细胞的中间丝。 DCX是表示在中间祖细胞，神经母细胞和未成熟的神经元微管相关蛋白。基于这三个标记和标记的细胞四个不同的祖细胞的亚型可以识别的形态特征的（共）表达：1型（GFAP +，巢蛋白+，DCX - ），2a型（GFAP - ，巢蛋白+ ，DCX - ），2B型（GFAP - ，+巢，DCX +）和3型（GFAP - ，巢- ，DCX +）1。 DCX用的NeuN，这表现在有丝分裂后的神经元，一起共同标签允许immatur分化E（DCX +，+的NeuN）和成熟（DCX - ，+的NeuN）颗粒神经元。 上面提到的标记物经常用于免疫荧光共标记和随后的共聚焦显微镜来分析新生细胞的数目和身份。这通常需要来自不同宿主物种的抗体，以防止不希望的抗体交叉反应性。然而，大多数适于神经发生研究初级抗体被升高或者家兔或小鼠（ 例如，小鼠α-BrdU的小鼠α-的NeuN，兔α-Ki67的，兔α-GFAP）。这导致以可能在一个单一的切片进行评估抗原的数量和组合严重的局限性。这反过来不仅增加了染色的努力，因为多种染色必须执行，而且还可能影响结果的可靠性。此外，一些抗原易受福尔马林固定诱导抗原掩蔽（ 例如 Ki67的，巢）。我们在此从古典单和多协议免疫标记描述修改（ 如表位检索，多个连续染色，使用巢蛋白GFP转基因小鼠12）克服许多问题。特别是，连续的多个免疫协议允许对染色即使抗体的一部分从同一主机衍生多达四个不同的抗原。这使得能够同时检测1型，图2a，2b型和3型祖细胞，以及在一个单一的部分其增殖活性。

<table border="0" cellpadding="0" cellspacing="0" width="665">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:277px;">Name</td>
			<td style="width:147px;">Company</td>
			<td style="width:111px;">Catalog Number</td>
			<td style="width:131px;">Comments &#38; Dilutions</td>
		</tr>
		<tr height="13">
			<td height="13" style="height:13px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thymidine analog administration</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-Bromo-2&#8242;-deoxyuridine, BrdU</td>
			<td>Sigma-Aldrich</td>
			<td>B9285</td>
			<td rowspan="4" style="width:131px;">toxic (mutagenic, teratogenic)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-Chloro-2&#8242;-deoxyuridine, CldU</td>
			<td>Sigma-Aldrich</td>
			<td>C6891</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-Chloro-2&#8242;-deoxyuridine, CldU</td>
			<td>MP Biomedicals</td>
			<td>2105478</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5-Iodo-2&#8242;-deoxyuridine, IdU</td>
			<td>MP Biomedicals</td>
			<td>2100357</td>
		</tr>
		<tr height="10">
			<td height="10" style="height:11px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoflurane-Actavis</td>
			<td>Piramal Healthcare</td>
			<td>700211</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Paraformaldehyde powder (PFA)</td>
			<td>Riedel-De H&#228;en</td>
			<td>16005</td>
			<td>toxic, flammable</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Perfusion pump PD5206</td>
			<td>Heidolph Instruments</td>
			<td>523-52060-00</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Masterflex Tygon lab tubing, &#216; 0.8 mm&#160;</td>
			<td>Thermo Fischer Scientific</td>
			<td>06409-13</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Feeding needle, straight, 21G, 1.75mm olive tip, 40mm</td>
			<td>Agnthos</td>
			<td>1036</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Freezing microtome Microm HM 400</td>
			<td>Thermo Fischer Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">24 Well Cell Culture Multiwell Plates</td>
			<td style="width:147px;">Greiner Bio-One</td>
			<td>662160</td>
			<td></td>
		</tr>
		<tr height="10">
			<td height="10" style="height:11px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunohistochemistry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tefal Vitacuisine Steamer</td>
			<td>Tefal</td>
			<td>VS 4001</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:277px;">Netwell 24mm Polyester Mesh Membrane Inserts Pre-Loaded in 6-Well Culture Plates</td>
			<td>Corning</td>
			<td>3479</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:277px;">Netwell 15mm Polyester Mesh Membrane Inserts Pre-Loaded in 12-Well Culture Plates</td>
			<td>Corning</td>
			<td>3477</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:277px;">Netwell Plastic 6-Well Carrier Kit for 24mm Polyester Mesh Membrane Inserts</td>
			<td>Corning</td>
			<td>3521</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:277px;">Netwell Plastic 12-Well Carrier Kit for 15mm Polyester Mesh Membrane Inserts</td>
			<td>Corning</td>
			<td>3520</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vectastain Elite ABC Kit</td>
			<td>Vector Laboratories</td>
			<td>PK-6100</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:277px;">DAB (3,3&#8242;-Diaminobenzidine tetrahydrochloride hydrate)</td>
			<td>Sigma-Aldrich</td>
			<td>D-5637</td>
			<td style="width:131px;">carcinogenic, light sensitive</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Fluoromount-G</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;">rabbit IgG1 &#945;-Ki67</td>
			<td style="width:147px;">Novocastra/ Leica Biosystems</td>
			<td>NCL-L-Ki67MM1</td>
			<td style="width:131px;">DAB 1:400/IF 1:100; requires epitope retrieval</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rabbit &#945;-GFAP, AS-3-GF</td>
			<td>Synaptic Systems</td>
			<td>173 002</td>
			<td>1:500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">goat IgG (H+L) &#945;-GFP</td>
			<td>Acris Antibodies</td>
			<td>R1091P</td>
			<td>1:300</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">mouse IgG1 &#945;-nestin</td>
			<td>Abcam</td>
			<td>ab6142</td>
			<td style="width:131px;">1:200; requires epitope retrieval</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">guinea pig IgG (H+L) &#945;-Doublecortin</td>
			<td>Merck Millipore</td>
			<td>AB2253</td>
			<td>1:500</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">rat IgG2a &#945;-BrdU (ascites)</td>
			<td>AbD Serotec/ Bio-Rad</td>
			<td>OBT0030CX</td>
			<td style="width:131px;">for detection of BrdU; DAB 1:500/IF 1:400</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;">rat IgG2a &#945;-BrdU (purified)</td>
			<td>AbD Serotec/ Bio-Rad</td>
			<td>OBT0030&#160;&#160;</td>
			<td style="width:131px;">for detection of CldU; DAB 1:500/IF 1:250-400</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;">mouse IgG1&#312; &#945;-BrdU</td>
			<td>BD Biosciences</td>
			<td>347580</td>
			<td style="width:131px;">for detection of IdU; DAB 1:500/IF 1:350</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mouse IgG1 &#945;-NeuN</td>
			<td>Merck Millipore</td>
			<td>MAB377</td>
			<td>1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">donkey &#945;-guinea pig IgG (H+L)-Biotin</td>
			<td>Dianova</td>
			<td>711-065-152</td>
			<td>1:500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">donkey &#945;-rat IgG (H+L)-Biotin</td>
			<td>Dianova</td>
			<td>712-065-150</td>
			<td>1:500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">donkey &#945;-mouse IgG (H+L)-Biotin</td>
			<td>Dianova</td>
			<td>715-065-151</td>
			<td>1:500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">goat &#945;-rat IgG (H+L)-Alexa Fluor 488</td>
			<td>Molecular Probes</td>
			<td>A11006</td>
			<td>1:250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">donkey &#945;-goat IgG (H+L)-Alexa Fluor 488</td>
			<td>Molecular Probes</td>
			<td>A11055</td>
			<td>1:250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">donkey &#945;-mouse IgG (H+L)-FITC, Fab-Fragment</td>
			<td>Dianova</td>
			<td>715-097-003</td>
			<td>1:100</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">donkey &#945;-mouse IgG (H+L)-Alexa Fluor 647</td>
			<td>Dianova</td>
			<td>715-605-151</td>
			<td>1:250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">donkey &#945;-guinea pig IgG (H+L)-Alexa Fluor 647</td>
			<td>Dianova</td>
			<td>706-605-148</td>
			<td>1:250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">donkey &#945;-rat IgG (H+L)-Rhodamine Red-X</td>
			<td>Dianova</td>
			<td>712-295-150</td>
			<td>1:250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">donkey &#945;-rabbit IgG (H+L)-Rhodamine Red-X</td>
			<td>Dianova</td>
			<td>711-295-152</td>
			<td>1:250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">donkey &#945;-guinea pig IgG (H+L)-Rhodamine Red-X</td>
			<td>Dianova</td>
			<td>706-296-148</td>
			<td>1:250</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptavidin-Rhodamine Red-X&#160;</td>
			<td>Dianova</td>
			<td>016-290-084</td>
			<td>1:500</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">goat &#945;-rabbit IgG (H+L)-AMCA</td>
			<td>Dianova</td>
			<td>111-155-144</td>
			<td style="width:131px;">1:250, works only with rabbit &#945;-GFAP</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hoechst 33342</td>
			<td>Molecular Probes</td>
			<td>H3570</td>
			<td>1:1000</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td>Molecular Probes</td>
			<td>D1306</td>
			<td>1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blocking</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fab-fragment donkey &#945;-mouse IgG (H+L)</td>
			<td>Dianova</td>
			<td>715-007-003</td>
			<td>1:20</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fab-fragment donkey &#945;-rabbit IgG (H+L)</td>
			<td>Dianova</td>
			<td>711-007-003</td>
			<td>1:20</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal donkey serum</td>
			<td>Merck Millipore</td>
			<td>S30</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal rabbit serum</td>
			<td>Dianova</td>
			<td>011-000-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Normal goat serum</td>
			<td>Dianova</td>
			<td>005-000-001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine Serum Albumine</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Histology</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Cresyl violett</td>
			<td>Sigma-Aldrich</td>
			<td>C5042</td>
			<td></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:277px;">Neo-Clear</td>
			<td>Merck Millipore</td>
			<td>109843</td>
			<td style="width:131px;">non-toxic xylene substitute</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:277px;">Neo-Mount</td>
			<td>Merck Millipore</td>
			<td>109016</td>
			<td style="width:131px;">permanent mounting medium</td>
		</tr>
		<tr height="10">
			<td height="10" style="height:11px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscopy</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">Axioskop 2</td>
			<td>Carl Zeiss Microscopy</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:277px;">LSM 710</td>
			<td>Carl Zeiss Microscopy</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

immunohistochemistry and multiple labeling with antibodies from the same host species to study adult hippocampal neurogenesis

成人神经发生是在其从激活的神经干细胞通过越来越致力于中间祖亚型产生新的神经元一高度调节，多阶段的过程。每个这些亚型的表达的一组，连同具体形态学标准，可以用于其识别特定的分子标志物。典型地，免疫荧光技术应用于涉及亚型特异性抗体结合外切或内源性增殖标记。在此我们描述免疫标记成人海马神经发生的各个阶段的检测和定量的方法。这些包括胸苷类似物，穿心灌注，组织处理，热诱导抗原活化，ABC免疫组织化学，多个间接免疫荧光，激光共聚焦显微镜和细胞量化的应用。此外我们提出了一个连续的多个免疫协议，规避问题ü所产生的由使用在相同的宿主物种中产生的第一抗体的需要sually。它允许一个准确的识别所有海马祖亚型连同在一个单一的部分扩散标记。这些技术是一种强大的工具，研究不同祖亚型并联的调节，它们在脑病理学的参与以及它们在特定大脑功能的作用。

我们采用上述量化和在产后和成年海马表征新生细胞的方法。因此，我们用野生型和神经缺陷型细胞周期蛋白D2敲出收纳已知影响神经发生的速率（ 即，丰富的环境中，EE）13,14的条件下（CCND2 KO）小鼠。对任何Ki67的，尿嘧啶，CldU或IDU免疫组化染色DAB一贯揭示野生型和CCND2 KO小鼠（ 图3）15之间的新生细胞数量的差异。此外，随着CldU和IDU连续注射等?...

Watch this Scientific Journal Video about Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis at JoVE.com

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

免疫组化和多标签与同一宿主物种抗体研究成人神经发生

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

immunohistochemistry-multiple-labeling-with-antibodies-from-same-host

Research

JoVE Journal

Neuroscience

26.4K 次观看.  Jena University Hospital. This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

视频: Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

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

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

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

幼虫，蛹和成人的解剖和免疫组化<em&gt;果蝇</em&gt;视网膜

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

科研

神经科学

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

成人下丘脑神经发生局灶性放射性抑制功能审讯

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Traditional methods of immunohistochemistry (IHC) following tissue fixation allow visualization of various cell types. These typically proceed with the application of antibodies to bind antigens and identify cells with characteristics that are a function of the inherent biology and development. Adult hippocampal neurogenesis is a sequential process wherein a quiescent neural stem cell can become activated and proceed through stages of proliferation, differentiation, maturation and functional integration. Each phase is distinct with a characteristic morphology and upregulation of genes. Identification of these phases is important to understand the regulatory mechanisms at play and any alterations in this process that underlie the pathophysiology of debilitating disorders. Our heat-induced antigen retrieval approach improves the intensity of the signal that is detected and allows correct identification of the progenitor cell type. As discussed in this paper, it especially allows us to circumvent current problems in detection of certain progenitor cell types.

During adult hippocampal neurogenesis, a distinct set of genetic markers are expressed when a quiescent neural stem cell sequentially progresses and develops into a functionally integrated neuron in the circuit. Using heat-induced antigen retrieval, progenitor cell types that are otherwise difficult to detect are identified with improved effectiveness.

Traditional  methods of immunohistochemistry (IHC) following tissue fixation allow visualization  of various cell types. These typically proceed with the ...

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 ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

方法对NF-κB依赖的成年神经发生的调制与分析

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive functions including decision making, sensory-guided behavior or memory. To understand how such intricate neuronal networks perform such tasks, a crucial step is to determine the function (or electrical activity) of individual cell types within the network, preferentially when the animal is performing a relevant cognitive task. Additionally, it is equally important to determine the anatomical structure of the network and the morphological architecture of the individual neurons to allow reverse engineering the cortical network. Technical breakthroughs available today allow recording cellular activity in awake, behaving animals with the valuable option of post&#160;hoc identifying the recorded neurons. Here, we demonstrate the juxtasomal biocytin labeling technique, which involves recording action potential spiking in the extracellular (or loose-patch) configuration using conventional patch pipettes. The juxtasomal recording configuration is relatively stable and applicable across behavioral conditions, including anesthetized, sedated, awake head-fixed, and even in the freely moving animal. Thus, this method allows linking cell-type specific action potential spiking during animal behavior to reconstruction of the individual neurons and ultimately, the entire cortical microcircuit. In this video manuscript, we show how individual neurons in the juxtasomal configuration can be labeled with biocytin in the urethane-anaesthetized rat for post&#160;hoc identification and morphological reconstruction.

To understand the structure of neuronal networks, functional and morphological characterization of individual neurons is a necessity. Here, we demonstrate juxtasomal biocytin labeling, which allows electrophysiological recordings in the extracellular configuration, yet maintaining the ability to intracellularly label the neuron for post&#160;hoc reconstruction of dendritic and axonal architecture.

Juxtasomal生物胞素标记来研究单个皮层神经元的结构与功能的关系

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

斑马鱼成人端脑的一刀，受伤：一个方法调查脊椎动物脑部神经发​​生与再生

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

评估主要神经再生<em&gt;爪</em&gt;胚胎使用免疫染色

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.