Name: immunohistochemistry and multiple labeling with antibodies from the same host species to study adult hippocampal neurogenesis
Uploaded: 2015-04-22T13:00:00.000+00:00
Duration: 9 min 24 s
Description: 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

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>

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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，但浓度不同而有所不同的实验条件（高达300毫克/千克的快速浓注）8,16。如果IDU和CldU必须被顺序地对细胞群的颞歧视注​​入它是强制性的，以注入等摩尔浓度（ <em&gt;如，42.5毫克/公斤CldU和57.5毫克/公斤IDU）。需要注意的是胸苷类似物可能是有毒的，以分裂的细胞，并且还可以标记受损细胞经历DNA合成16。另一个缺点是，DNA变性所需的免疫组化检测胸苷类似物可能与抗体或其它DNA标记技术的抗原性干扰（ 例如，DAPI，Hoechst公司33258）。免疫组化检测内源性增殖标记Ki67的一样可能是一个适当的替代10,11。然而，这种只允许一个快照增殖活性在灌注时，追溯出生约会和命运的分析是不可能的。 对于免疫染色，切片通常在装有载体板6孔板加工自由浮动，通常是和网状刀片从而简化了从一个溶液到另一个其传输。作为例外，阻断，抗体孵育和ABC反应完成在12或24孔板无网状刀片节约昂贵的解决方案（0.5到1ml每孔是足够的，这取决于要被染色的切片的数量）。在这些步骤中，转移部分用细刷，需要改变的解决方案时，将清洗的帮助。防止干燥出部分和执行采取&gt; 1小时的腐殖化室孵化。所有温育都必须在连续搅拌（最大150rpm）下进行。始终测试和滴定每个新抗体不少。较长抗体孵育时间（2天在4°C）可以提高染色。在情况下，只小鼠的抗体可针对想要小鼠组织中进行可视化的抗原最好是阻断内源性免疫球蛋白与高度浓缩的单价Fab片段α - 小鼠（第3.4节）。每当使用这种技术延长了以下漂洗步骤。否则，未绑定的Fab片段可能会绑定到你的主鼠抗体。盐酸预处理和硼酸盐中和只要求进行检测的胸苷类似物。在12分钟的过氧化物酶反应导致一个最佳的信噪比与在我们的协议所施加的条件和抗体。正如任何酶反应，过氧化物酶反应的速率取决于多种因素（ 例如，温度，pH，酶和底物的浓度）。因此，反应时间需要任何新的抗体组进行优化。可视化的解剖结构在DAB污渍非常弱的背景下（ 例如，CldU），片可以counterstained。在一个非常微弱染液不损害特定DAB信号在部分中的甲酚紫方法中描述的结果3.5。 对于多个免疫，使用那些成长于不同的亚型不同物种或主要抗体，并按照3.2.1节。否则，执行连续多次免疫标记进行了优化，以检测多个主要抗体来自同一宿主物种（ 如小老鼠或兔子;第3.2.2节）。该方法的基本原理已发明了Ferguson和同事成功染色不同的肌肉的标记与两个鼠标初级抗体17谁。用于阻断在步骤3.2.2.5 Fab片段应来源于相同宿主物种作为二次抗体。在节3.2.2中描述的浓度和温育时间为本文所描述的特异性抗体的优化，并进行调整，对任何新的抗体的组合。要注意的第一抗体孵育的顺序可能强烈影响的结果（如在图7A中表示的）。只要适用，由从相同物种来源的初级抗体检测的抗原应该显示其简化闭锁不足的检测不同的表达模式。为了消除抗体的交叉反应或假阳性的可能是埃森TiAl基包括适当的管制（ 即只中学抗体控制;完整的免疫组化的第一抗原随后与第二原发抗体或单独第二二次抗体孵化）。18 图2B表明，在我们手中运作良好的抗体组合。对于多个免疫荧光也建议使用所提出的在同种（ 例如，驴）二次抗体和有最小的交叉反应性的组织和血清来自其它物种（ 例如，预吸附）蛋白。否则，第3.2.3节可能有助于防止任何意外的种间交叉反应。荧光染料选择以最小的光谱重叠适合您的检测系统（ 如 AMCA，的Alexa Fluor 488，罗丹明红，将Alexa Fluor 647）。如果一个核染液是必需的，DAPI或Hoechst公司33342（10微克/毫升）添加到该次级抗体混合物（以后，没有近紫外二级抗体可以是我们ED）。如果组织已经被预先处理，用HCl这些核counterstains不起作用。同样，HCl处理可能损害某些抗原的检测，因此，需要进行测试其对个人抗体的性能的影响。一旦荧光标记的二抗已应用于保护部分从光。 CldU和IDU可与两个不同的BrdU抗体的帮助，交叉反应的可视化或者与CldU（大鼠α-的BrdU）或IDU（鼠α-的BrdU）9,19。如果两个胸苷类似物已被注射到一只动物它是使用纯化的大鼠αBrdU抗体检测CldU因为非纯化的抗体的交叉反应，IDU绝对必要的。 9所述CldU信号需要被放大为免疫荧光共标记来实现等效的检测都胸苷类似物所描述的维加等人因此，与其荧光染料缀合的第二抗体（α-鼠）荧光染料共轭链霉添加以下孵化的生物素化的第二抗体（α-鼠;第3.2.4节）。排除任何不期望的抗体的交叉反应包括从接收到的任一或IDU CldU小鼠控制部（另请参阅图8）。为了验证等效检测从动物IDU和CldU使用部分即同时被注射CldU和IDU等摩尔量（参见图4）。 研究1型和2型祖细胞，我们优选使用巢蛋白-GFP转基因小鼠12为巢蛋白抗体常常没有提供令人满意的结果。巢蛋白-GFP小鼠具有在特定发育阶段可靠地可视巢蛋白阳性祖细胞和它们的形态特征的优点。将GFP-信号需要通过间接免疫荧光法用αGFP抗体进行放大。二次抗体应耦合到fluorochrOME具有相似于GFP的光谱特性。如果需要的话，巢蛋白-GFP小鼠可以杂交到其它突变体鼠标线，调查特定基因在神经发生中的参与。现在，改进的巢蛋白的抗体被提供哪些标签胞体以及过程，可以用来作为替代转基因的方法（见材料清单）。 关键是要遵守24小时固定，以防止overfixation。甲醛在组织中的化学反应伙伴主要是蛋白质和甲醛产生在其侧链导致交联的相邻氨基酸（通过亚甲基桥的形成）反应性羟甲基。这可能导致的抗原表位和免疫反应的损失掩蔽（参见材料清单评价特异性抗体）。要恢复丢失的免疫反应，几个表位检索方法存在通过电子邮件的突破甲醛引起的蛋白质交联键xposure加热20。在这个协议中，柠檬酸缓冲方法效果优于他人在产后和成年海马神经检测的（3.3节）。需要注意的是非常年轻的动物切片（&lt;P21）可能会变得非常脆弱或扭曲，需要特别小心处理。另外，表位的检索可能是有害的一些表位，因此，各个抗体应该测试它们是否适于多重染色需要此预处理。 总之，这个协议是有利于成人海马神经发生的分析基本和阐述的技术的集合。它特别允许明确识别所有海马祖亚连同从一个单一的部分扩散或出生约会标记。这些技术可以适用于抗体或其他物种的任何组合来研究海马组织的发展和调节PAL祖细胞响应于特定刺激和其在特定大脑功能的参与。

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><tbody><tr><td colspan="4">&lt;em&gt;Thymidine analog administration&lt;/em&gt;</td></tr><tr><td>5-Bromo-2&amp;prime;-deoxyuridine, BrdU</td><td>Sigma-Aldrich</td><td>B9285</td><td>toxic (mutagenic, teratogenic)</td></tr><tr><td>5-Chloro-2&amp;prime;-deoxyuridine, CldU</td><td>Sigma-Aldrich</td><td>C6891</td><td>toxic (mutagenic, teratogenic)</td></tr><tr><td>5-Chloro-2&amp;prime;-deoxyuridine, CldU</td><td>MP Biomedicals</td><td>2105478</td><td>toxic (mutagenic, teratogenic)</td></tr><tr><td>5-Iodo-2&amp;prime;-deoxyuridine, IdU</td><td>MP Biomedicals</td><td>2100357</td><td>toxic (mutagenic, teratogenic)</td></tr><tr><td colspan="4">&lt;em&gt;Tissue preparation&lt;/em&gt;</td></tr><tr><td>Isoflurane-Actavis</td><td>Piramal Healthcare</td><td>700211</td><td></td></tr><tr><td>Paraformaldehyde powder (PFA)</td><td>Riedel-De H&amp;auml;en</td><td>16005</td><td>toxic, flammable</td></tr><tr><td>Perfusion pump PD5206</td><td>Heidolph Instruments</td><td>523-52060-00</td><td></td></tr><tr><td>Masterflex Tygon lab tubing, &amp;Oslash; 0.8 mm</td><td>Thermo Fischer Scientific</td><td>06409-13</td><td></td></tr><tr><td>Feeding needle, straight, 21 G, 1.75 mm olive tip, 40 mm</td><td>Agnthos</td><td>1036</td><td></td></tr><tr><td>Freezing microtome Microm HM 400</td><td>Thermo Fischer Scientific</td><td></td><td></td></tr><tr><td>24-well Cell culture multiwell plates</td><td>Greiner Bio-One</td><td>662160</td><td></td></tr><tr><td colspan="4">&lt;em&gt;Immunohistochemistry&lt;/em&gt;</td></tr><tr><td>Tefal vitacuisine steamer</td><td>Tefal</td><td>VS 4001</td><td></td></tr><tr><td>Netwell 24 mm polyester mesh membrane inserts</td><td>Corning</td><td>3479</td><td>pre-loaded in 6-well culture plates</td></tr><tr><td>Netwell 15 mm polyester mesh membrane inserts</td><td>Corning</td><td>3477</td><td>pre-loaded in 12-well culture plates</td></tr><tr><td>Netwell plastic 6-well carrier kit</td><td>Corning</td><td>3521</td><td>for 24 mm polyester mesh membrane inserts</td></tr><tr><td>Netwell plastic 12-well carrier kit</td><td>Corning</td><td>3520</td><td>for 15 mm polyester mesh membrane inserts</td></tr><tr><td>Vectastain Elite ABC kit</td><td>Vector Laboratories</td><td>PK-6100</td><td></td></tr><tr><td>DAB (3,3&amp;prime;-Diaminobenzidine tetrahydrochloride hydrate)</td><td>Sigma-Aldrich</td><td>D-5637</td><td>carcinogenic, light sensitive</td></tr><tr><td>Fluoromount-G</td><td>SouthernBiotech</td><td>0100-01</td><td></td></tr><tr><td colspan="4">&lt;em&gt;Primary antibodies&lt;/em&gt;</td></tr><tr><td>Rabbit IgG1 &amp;alpha;-Ki67</td><td>Novocastra/ Leica Biosystems</td><td>NCL-L-Ki67MM1</td><td>DAB 1:400/IF 1:100; requires epitope retrieval</td></tr><tr><td>Rabbit &amp;alpha;-GFAP, AS-3-GF</td><td>Synaptic Systems</td><td>173 002</td><td>1:500</td></tr><tr><td>Goat IgG (H+L) &amp;alpha;-GFP</td><td>Acris Antibodies</td><td>R1091P</td><td>1:300</td></tr><tr><td>Mouse IgG1 &amp;alpha;-nestin</td><td>Abcam</td><td>ab6142</td><td>1:200; requires epitope retrieval</td></tr><tr><td>Guinea pig IgG (H+L) &amp;alpha;-Doublecortin</td><td>Merck Millipore</td><td>AB2253</td><td>1:500</td></tr><tr><td>Rat IgG2a &amp;alpha;-BrdU (ascites)</td><td>AbD Serotec/ Bio-Rad</td><td>OBT0030CX</td><td>for detection of BrdU; DAB 1:500/IF 1:400</td></tr><tr><td>Rat IgG2a &amp;alpha;-BrdU (purified)</td><td>AbD Serotec/ Bio-Rad</td><td>OBT0030&amp;nbsp;&amp;nbsp;</td><td>for detection of CldU; DAB 1:500/IF 1:250-400</td></tr><tr><td>Mouse IgG1 &amp;alpha;-NeuN</td><td>Merck Millipore</td><td>MAB377</td><td>1:500</td></tr><tr><td>mouse IgG1&amp;#954; &amp;#945;-BrdU</td><td>BD Biosciences</td><td>347580</td><td>For detection of IdU; DAB 1:500/IF 1:350</td></tr><tr><td colspan="4">&lt;em&gt;Secondary antibodies&lt;/em&gt;</td></tr><tr><td>Donkey &amp;alpha;-guinea pig IgG (H+L)-Biotin</td><td>Dianova</td><td>711-065-152</td><td>1:500</td></tr><tr><td>Donkey &amp;alpha;-rat IgG (H+L)-Biotin</td><td>Dianova</td><td>712-065-150</td><td>1:500</td></tr><tr><td>Donkey &amp;alpha;-mouse IgG (H+L)-Biotin</td><td>Dianova</td><td>715-065-151</td><td>1:500</td></tr><tr><td>Goat &amp;alpha;-rat IgG (H+L)-Alexa Fluor 488</td><td>Molecular Probes</td><td>A11006</td><td>1:250</td></tr><tr><td>Donkey &amp;alpha;-goat IgG (H+L)-Alexa Fluor 488</td><td>Molecular Probes</td><td>A11055</td><td>1:250</td></tr><tr><td>Donkey &amp;alpha;-mouse IgG (H+L)-FITC, Fab-Fragment</td><td>Dianova</td><td>715-097-003</td><td>1:100</td></tr><tr><td>Donkey &amp;alpha;-mouse IgG (H+L)-Alexa Fluor 647</td><td>Dianova</td><td>715-605-151</td><td>1:250</td></tr><tr><td>Donkey &amp;alpha;-guinea pig IgG (H+L)-Alexa Fluor 647</td><td>Dianova</td><td>706-605-148</td><td>1:250</td></tr><tr><td>Donkey &amp;alpha;-rat IgG (H+L)-Rhodamine Red-X</td><td>Dianova</td><td>712-295-150</td><td>1:250</td></tr><tr><td>Donkey &amp;alpha;-rabbit IgG (H+L)-Rhodamine Red-X</td><td>Dianova</td><td>711-295-152</td><td>1:250</td></tr><tr><td>Donkey &amp;alpha;-guinea pig IgG (H+L)-Rhodamine Red-X</td><td>Dianova</td><td>706-296-148</td><td>1:250</td></tr><tr><td>Streptavidin-rhodamine Red-X</td><td>Dianova</td><td>016-290-084</td><td>1:500</td></tr><tr><td>Goat &amp;alpha;-rabbit IgG (H+L)-AMCA</td><td>Dianova</td><td>111-155-144</td><td>1:250, works only with rabbit &amp;alpha;-GFAP</td></tr><tr><td>Hoechst 33342</td><td>Molecular Probes</td><td>H3570</td><td>1:1000</td></tr><tr><td>DAPI</td><td>Molecular Probes</td><td>D1306</td><td>1:1000</td></tr><tr><td colspan="4">&lt;em&gt;Blocking&lt;/em&gt;</td></tr><tr><td>Fab-fragment donkey &amp;alpha;-mouse IgG (H+L)</td><td>Dianova</td><td>715-007-003</td><td>1:20</td></tr><tr><td>Fab-fragment donkey &amp;alpha;-rabbit IgG (H+L)</td><td>Dianova</td><td>711-007-003</td><td>1:20</td></tr><tr><td>Normal donkey serum</td><td>Merck Millipore</td><td>S30</td><td></td></tr><tr><td>Normal rabbit serum</td><td>Dianova</td><td>011-000-010</td><td></td></tr><tr><td>Normal goat serum</td><td>Dianova</td><td>005-000-001</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td colspan="4">&lt;em&gt;Histology&lt;/em&gt;</td></tr><tr><td>Cresyl violet</td><td>Sigma-Aldrich</td><td>C5042</td><td></td></tr><tr><td>Neo-Clear</td><td>Merck Millipore</td><td>109843</td><td>non-toxic xylene substitute</td></tr><tr><td>Neo-Mount</td><td>Merck Millipore</td><td>109016</td><td>permanent mounting medium</td></tr><tr><td colspan="4">&lt;em&gt;Microscopy&lt;/em&gt;</td></tr><tr><td>Axioskop 2</td><td>Carl Zeiss Microscopy</td><td></td><td></td></tr><tr><td>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连续注射等摩尔我们能够区分在EE（ 图3C，D）的特定时期出生的细胞群。采用第3.2.4我们可以成功检测等信号强度和CldU和IDU他们同时交付（ 图4）后，优秀的重叠。的BrdU标记新生细胞可以与斯坦进行表型准免疫荧光法通过或者同时施加的BrdU和的NeuN，（ 图5）或尿嘧啶，GFAP，巢蛋白-GFP和DCX抗体（ 图6B）。这种技术允许成功鉴定1型，2a，2b和一个单一的样品（ 图6B中的D）在3个祖细胞。与祖标记共同标记Ki67的所需抗体的顺序应用作为对Ki67和GFAP的一抗在兔中提出（适用于第3.2.2节），而且第一抗体之一（山羊α-GFP）的制造中同一台主机作为辅助抗体之一（AMCA连词。山羊α-兔）。部分3.2.2和3.2.3的完美结合工作，如果切片中Ki67的第一培养与巢蛋白GFP抗体，其次在GFAP抗体，而不是相反（ 图6C）。图2B总结了批准的申请方案。 图2.连续多次免疫与来自同一主机物种的主要抗体。（A）示意图。（B）抗体及其免疫中按时间顺序的成功组合。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52551/52551fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52551/52551fig3.jpg" /> ABC-免疫组化图3.光显微镜图像的新生细胞定量齿状回，描绘的是在WT不同的增殖标记和神经缺陷CCND2 KO小鼠100X和400X放大倍数的比较。（A） </stron<html> g&gt;的Ki67的-DAB染色显示集群在齿状回（产后35天）的颗粒下区增殖细胞。增殖细胞的数目减少在相比WT小鼠CCND2 KO小鼠。（B）的新生细胞的28天的BrdU给药（以上图像注射方案）确认降低增殖率CCND2 KO小鼠后（℃）CldU给药在EE（上面的图片注入方案）的第6周透露WT的齿状回增加新生细胞的数量，但在EE的第8周未CCND2 KO小鼠（D）IDU-政府表现出类似的结果。比例尺代表50微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52551/52551fig3highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> fig4.jpg"/&gt; 同时传送等摩尔浓度后图4.免疫荧光共染色CldU和注射吸毒者。CldU标记细胞的信号放大导致CldU和IDU的优秀重叠。比例尺代表20微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52551/52551fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/52551/52551fig5.jpg" /> 图5.免疫联合标识的NeuN和BrdU的。 （A）中的BrdU施用6次，每8小时，连续2天开始，在出生后一天处死14.动物28天。（B）中与成熟的神经元标记物的增殖细胞的免疫荧光共标记的NeuN显示增殖数量减少细胞CCND2 KO与W相比牛逼的动物。比例尺条为50微米。（C）的 （B）的放大视图，示出在两个基因型共标记的细胞。比例尺条为20微米。（D）中 ，在齿状回一个的BrdU /的NeuN共标记的细胞的高倍率例子。比例尺代表10微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52551/52551fig5highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/52551/52551fig6.jpg" /> 图6.四免疫荧光共标记的不同增殖标志物GFAP，巢蛋白-GFP和DCX在巢蛋白-GFP的小鼠的齿状回表型分型初生颗粒细胞。（A）中的BrdU施用3次，每次2小时，在出生后70天。动物处死2小时后。共染色的BrdU，GFAP（B）的代表性图像，巢蛋白-GFP和四免疫荧光为Ki67蛋白，GFAP，巢蛋白-GFP和DCX的DCX。（C）的代表性的图像。（D）中 ，根据它们的免疫荧光标记的祖细胞类型的分类。基于该共表达不同的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选择: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 +）。比例尺条为20微米。（E）的成人海马神经发生的不同阶段的示意图概述。它示出了4祖细胞类型，未成熟的神经元和成熟粒细胞中，标志物的它们的特征的表达和它们的增殖能力。 ML - 分子细胞层，GCL -颗粒细胞层，SGL -颗粒下细胞层<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52551/52551fig6highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/52551/52551fig7.jpg" /> 图7.成功和非成功的连续染色与在相同物种中产生，这取决于抗体孵育的序列中的两个初级抗体实施例的共焦显微照片示出了连续的共染色针对Ki67和GFAP与初级抗体的结果都源自兔子。在（A）免疫组化法第一完成GFAP其次是染色Ki67的反对而导致两个信号的明显重叠。特别是Ki67的信号是非典型和类似了GFAP染色的信号。（B）表示从反向的结果与Ki67染色染色顺序完成对GFAP第一和随后的染色。这里，对于这两种抗原的信号相似的预期表达模式:Ki67的信号被限制在颗粒下区中的细胞核而GFAP信号被发现细胞质，主要是在从所述颗粒下区产生的细胞过程。比例尺条为20微米。 RHX -罗丹明X. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52551/52551fig7highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图8" src="/files/ftp_upload/52551/52551fig8.jpg" /> 图8.特异性检测CldU和IDU。小鼠α-BrdU的抗体注射或者与CldU或IDU（非同时应用程序）。在（A）CldU（A1）或IDU（A2）的脑切片注入小鼠IMMUnostained使用纯化的大鼠αBrdU抗体应特异性交叉反应，以CldU，但不能IDU。（B）表示从CldU（B1）或IDU（B2）染色大脑结果注射小鼠的小鼠α-BrdU的抗体，预计将用IDU交叉反应，但不CldU。比例尺代表20微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52551/52551fig8highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

26.5K 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

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

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

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

嵌入后突触蛋白的免疫标记海马切片培养

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

科研

神经科学

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 ...

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

引物对免疫冰冻切片大鼠脑组织:例为染色小胶质细胞和神经元

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we describe a pERK immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. Specifically, we used pERK immunostaining to study neuronal activation following Morris water maze (MWM, a classical hippocampal-dependent learning task) in Engrailed-2 knockout (En2-/-) mice, a model of autism spectrum disorders (ASD). As compared to wild-type (WT) controls, En2-/- mice showed significant spatial learning deficits in the MWM. After MWM, significant differences in the number of pERK-positive neurons were detected in specific hippocampal subfields of En2-/- mice, as compared to WT animals. Thus, our protocol can robustly detect differences in pERK-positive neurons associated to hippocampal-dependent learning impairment in a mouse model of ASD. More generally, our protocol can be applied to investigate the profile of hippocampal neuron activation in both genetic or pharmacological mouse models characterized by cognitive deficits.

We describe an immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. This protocol can be applied to both genetic or pharmacological mouse models characterized by cognitive deficits.

海马神经元活动的可视化免疫组化空间后学习神经发育障碍的小鼠模型

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

成人希马神经前体细胞的乳化电路特定调节

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate ...

Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to obtain commercial monoclonal or polyclonal antibodies that recognize specific cell structures and proteins in parasites. Besides, there are few commercial antibodies available to label trypanosomatids. Usually, polyclonal antibodies against parasites are prepared in-house and could be more challenging to use in combination with other antibodies produced in the same species. Here, the protocol demonstrates how to use polyclonal and monoclonal antibodies raised in the same species to perform double labeling immunofluorescence to study host cell and pathogen interactions. To achieve the double labeling immunofluorescence, it is crucial to incubate first the mouse polyclonal antibody and then follow the incubation with the secondary mouse IgG antibody conjugated to any fluorochrome. After that, an additional blocking step is necessary to prevent any trace of the primary antibody from being recognized by the next secondary antibody. Then, a mouse monoclonal antibody and its specific IgG subclass secondary antibody conjugated to a different fluorochrome are added to the sample at the appropriate times. Additionally, it is possible to perform triple labeling immunofluorescence using a third antibody raised in a different species. Also, structures such as nuclei and actin can be stained subsequently with their specific compounds or labels. Thus, these approaches presented here can be adjusted for any cell whose sources of primary antibodies are limited.

Here, the protocol describes how to perform double labeling immunofluorescence using primary antibodies raised in the same species to study host-pathogen interactions. Also, it can include the third antibody from a different host in this protocol. This approach can be made in any cell type and pathogens.

使用来自同一物种的抗体进行双标记免疫荧光研究宿主 - 病原体相互作用

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to ...

免疫与感染

Fluorescence-Activated Nuclei Negative Sorting of Neurons Combined with Single Nuclei RNA Sequencing to Study the Hippocampal Neurogenic Niche

Adult Hippocampal Neurogenesis (AHN), which consists of a lifelong maintenance of proliferative and quiescent neural stem cells (NSCs) within the sub-granular zone (SGZ) of the dentate gyrus (DG) and their differentiation from newly born neurons into granule cells in the granule cell layer, is well validated across numerous studies. Using genetically modified animals, particularly rodents, is a valuable tool to investigate signaling pathways regulating AHN and to study the role of each cell type that compose the hippocampal neurogenic niche. To address the latter, methods combining single nuclei isolation with next generation sequencing have had a significant impact in the field of AHN to identify gene signatures for each cell population. Further refinement of these techniques is however needed to phenotypically profile rarer cell populations within the DG. Here, we present a method that utilizes Fluorescence Activated Nuclei Sorting (FANS) to exclude most neuronal populations from a single nuclei suspension isolated from freshly dissected DG, by selecting unstained nuclei for the NeuN antigen, in order to perform single nuclei RNA sequencing (snRNA-seq). This method is a potential steppingstone to further investigate intercellular regulation of the AHN and to uncover novel cellular markers and mechanisms across species.

Presented here is a method to sequence single nuclei isolated from the mouse dentate gyrus that excludes most neurons through fluorescence-activated nuclei (FAN)-sorting. This approach generates high-quality expression profiles and facilitates the study of most other cell types represented in the niche, including scarce populations such as neural stem cells.

荧光激活神经元核负分选结合单核RNA测序研究海马神经源性生态位

Adult Hippocampal Neurogenesis (AHN), which consists of a lifelong maintenance of proliferative and quiescent neural stem cells (NSCs) within the ...

Immunohistochemistry Techniques to Analyze Cellular Proliferation and Neurogenesis in Rats Using the Thymidine Analog BrdU

One of the most important things in the field of adult hippocampal neurogenesis (AHN) is the identification of the newly generated cells. The immunodetection of thymidine analogs (such as 5-Bromo-2&#39;-deoxyuridine (BrdU)) is a standard technique used for visualizing these newly generated cells. Therefore, BrdU is usually injected in small animals intraperitoneally, so the thymidine analog gets incorporated into dividing cells during DNA synthesis. Detection is performed by immunohistochemical analysis of brain slices. Every research group that has been using this technique can appreciate that it requires special attention to minute details to achieve a successful stain. For instance, an important step is DNA denaturation with HCl, which allows it to reach the cell nucleus to stain it. However, the existing scientific reports describe very few of such steps in detail. Therefore, standardizing the technique is challenging for new laboratories as it can take several months to yield positive and successful outcomes. The purpose of this work is to describe and elaborate the steps to obtain positive and successful outcomes of the immunostaining technique in detail when working with the thymidine analog BrdU. The protocol includes the reagent preparation and setup, administration of thymidine analog in a rodent, transcardial perfusion, tissue preparation, peroxidase immunohistochemical reaction, use of avidin-biotin complex, immunofluorescence, counterstaining, microscopy imaging, and cell analysis.

This paper presents four of the most common techniques for visualizing BrdU positive cells to measure adult neurogenesis in rats. This work includes instructions for reagent preparation, thymidine analog administration, transcardial perfusion, tissue preparation, peroxidase immunohistochemical reaction, immunofluorescence, signal amplification, counterstaining, microscopy imaging, and cell analysis.

使用胸苷类似物BrdU分析大鼠细胞增殖和神经发生的免疫组织化学技术

One of the most important things in the field of adult hippocampal neurogenesis (AHN) is the identification of the newly generated cells. The ...

生物学

Immunofluorescence Staining for Visualizing Proliferated Neural Stem Cells in Mouse Brain Sections

Source: Quintanilla, L. J., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/59237">Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells.</a> J. Vis. Exp. (2019)
This video demonstrates the procedure for staining newly proliferated neural stem cells in brain tissue sections using EdU incorporation, followed by immunofluorescence to visualize specific proteins, enhancing the understanding of cell dynamics and proliferation.

免疫荧光染色用于观察小鼠脑切片中增殖的神经干细胞

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Immunohistochemistry

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JoVE Encyclopedia of Experiments

Neuropathology

Developmental and Pediatric Disorders

An Immunofluorescence Staining Technique to Detect Adult Hippocampal Neurogenesis

Source: Ansorg, A., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/52551">Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis.</a> J. Vis. Exp. (2015)
The video illustrates an immunofluorescence staining method used to examine neurogenesis in the adult hippocampus. Initially, a mouse brain section undergoes treatment with primary antibodies directed at marker proteins associated with neurogenesis in neuronal progenitor cells. Next, it is labeled with secondary antibodies tagged with fluorophores. The brain section is then observed under a confocal microscope to distinguish various subtypes of progenitor cells, based on the expression patterns of distinct combinations of marker proteins.

一种检测成人海马神经发生的免疫荧光染色技术

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Tissue ...