我们感谢在科罗拉多大学博尔德分校教授迈克尔·Klymkowsky和南希Papalopulu教授在曼彻斯特大学的好心提供抗SOX3和抗MYT1抗体，分别为。我们感谢Papalopulu实验室的成员在发展这个协议的有见地的建议。这项工作是由两个治疗基金会助学金，以深圳和JL支持，从治疗基金会SZ / EA和JL / EA分别从威康信托基金会，以EA（WT082450MA）一个程序拨款，一个机构的战略支持授权二级项目资助从威康信托[097820 / Z / 11 / Z]。

所有的动物实验是从曼彻斯特动物福利中心的大学批准，由英国内政部项目许可都淹没了。 1.收集爪蟾胚胎的固定和<ol><li>准备实验试剂和材料。 <ol><li>制备56.5克氯化钠溶解在大约800毫升超纯水和加入1M的KCl储备溶液，1M 硫酸镁 ，1M 氯化钙 ，和1M HEPES pH 7.4的10倍Marc的改性林格（MMR），以实现最终浓度为20mM的KCl，10mM的硫酸镁 ，20mM的氯化钙 ，50mM的HEPES。由10 M氢氧化钠调节pH至7.4，然后调整最终体积至1L </li><li>消毒通过高压灭菌器在121℃，20分钟，在液体循环中的10倍的MMR溶液。在使用时，与DDH 2 O的稀释至0.1×终浓度，并加入20毫克/升庆大霉素的，以抑制微生物的生长。 </li><li>通过混合24克的Tris-HCl，5.6克亚磷酸三-b请10X TBS解决方案酶，88克NaCl和溶解在约900毫升超纯水。最终的解决方案将有大约7.6的pH值。与任一10 M氢氧化钠或浓HCl调整以达到7.6的最终pH和终体积为1L。 </li><li>使用后，稀释10倍与9份超纯水的TBS液1份做1X TBS。 </li><li>通过在约800毫升超纯水中溶解209.2克MOPS并加入0.5M的EGTA和1M MgSO 4上的储液来实现的20mM EGTA，10mM的硫酸镁的最终浓度使10×MEM盐。由10 M氢氧化钠调节pH至7.4，然后调整最终体积至1L </li><li>消毒通过高压灭菌器在121℃，20分钟的MEM盐溶液上的液体循环（该溶液可通过存储的几个月在常温下变黄或已经高压灭菌后，但这种变化的颜色，不影响其使用）。但是，不要使用长期储存（超过6个月）后的溶液。 </li><li>让1X MEM通过稀释的MEM盐类的1份，37％甲醛的1份与8份超纯水的（体积/体积，在4℃下稳定至少1-2周）的FA溶液。 </li><li>由4多聚甲醛粉末溶解在100毫升1×TBS溶液热的溶液到60℃制备的TBS 4％多聚甲醛（用于涉及毒伞素后续染色），并添加10 M氢氧化钠几滴协助溶解。等分试样在5-10毫升体积并冷冻在-20℃。不要重新冻结一旦解冻。 注意:多聚甲醛粉是一种刺激物，如果吸入，因此称重步骤应在通风橱中进行是有毒的。 </li><li>要与采样前螺旋瓶盖标签尽可能多的4毫升玻璃小瓶。 </li><li>通过倾倒20毫升40％鱼明胶（预热，在50℃的水浴）到50毫升离心管中制备15％明胶/ 15％蔗糖。加入8克蔗糖和填充管与1X TBS 50毫升刻度线。 </li><li>将明胶管上的旋转式混合机或滚床在室温下混合过夜。此明胶溶液是在4℃下1周是稳定的。不要使用过期的解决方案，不冻融。 </li></ol></li><li>准备和修复X.蟾或X.热带胚胎培养到需要的阶段。 <ol><li>文化受精X.蟾或X.热带胚胎0.1倍MMR与庆大霉素，直到所需的阶段。 注:一般情况下，收集阶段23和胚胎的40后来收集的胚胎阶段40后是可能的，在观察从脊髓轴突生长尤其是当，但请记住，更多的明胶渗透时间可能是必需的。胚胎可以是野生型，转基因，突变体，抑制剂治疗的，吗啉代（MO）-injected，或电穿孔23,24。 </li><li>收集20-50胚胎每次4毫升的玻璃小瓶中，尽量恢复介质可能与MEMFA替换。 注意:如果后续染色涉及毒伞素，用4％多聚甲醛代替MEMFA自商业提供的甲醛溶液通常含有高达10％的甲醇作为稳定剂将与鬼笔环肽染色干扰。 </li><li>在4℃固定过夜，或者，如果在一个紧迫性，在室温下在旋转混合器2小时。胚胎将在固定溶液稳定至少1周4℃。 </li><li>固定后，用1×TBS用0.05％的Triton X-100洗胚胎3次20分钟。最后一次洗涤后，除去尽可能多的TBS-的Triton尽可能并添加3毫升15％明胶/ 15％的蔗糖到每个小瓶中。 </li><li>放置小瓶在室温下床过夜的辊。比阶段40岁的胚胎，使用的渗透时间至少24小时。渗透后，立即进行第二天第2.2节。 </li></ol></li></ol> 2.安装和爪蟾胚胎Cryosectioning <ol><li>准备实验试剂和材料。 <ol><li>采取积极临时代办一箱编辑幻灯片，理想未开封。 注意:如果打开，保持在干燥状态下滑动（如干燥箱中）和1个月内使用，以确保在幻灯片上的静电荷被维持。不要使用过期的幻灯片，因为样品将在免疫脱落。 </li><li>预冷却低温恒温室-30℃。设置仪器参数，-35°C用于切片和12微米厚度的部分。安装厚玻璃盖片搬上了舞台，让低温恒温器平衡至少30分钟一节开始之前。 </li><li>准备画刷移动部分带，保持低温恒温箱内。 </li><li>准备铅笔在玻片上写，把它们在室温下。 </li><li>冷冻切片时戴上手套。不要用裸露的双手。 </li></ol></li><li>安装爪蟾胚胎<ol><li>小心吸5-10胚胎出用塑料或玻璃吸管的玻璃小瓶的而不引入气泡。胚胎转移中到安装腔室，并观察一个立体镜之下。填满用明胶溶液的安装腔，以确保部分块的刚性。 </li><li>安排胚胎按照图1采用一对细尖镊子。通过使用低温兼容的标记在腔室的边缘绘制的箭头标记的头的取向。对于胚胎的多个组，记下各组在相应的储藏室边的描述为好。 </li><li>小心地水平放置室在泡沫箱半满的干冰，并盖上盖子。观察5-10分钟安装室冻结。处理每个腔室串联（ 即放置先前室到干冰继续到下一个之前），因为这将留下足够的时间为每个腔室冻结并防止干冰框变得拥挤。 注:冷冻安装腔不需要水平放置，并且可以堆叠起来箱内。 </li><li>与cryosectioning进行或，如果需要的话，保持冷冻的样品在-80℃下为至少1-2周，而不会失去免疫原性。 </li></ol></li><li>安装爪蟾胚胎的冷冻切片<ol><li>按使用钝棒的腔室的底部（如铅笔）从室的冷冻样品块。 </li><li>加组织冷冻介质的数滴，（含醇-聚乙二醇和聚乙烯基介质）到样品保持盘并与胚胎的前端安装样品块指向上（ 图2）。允许mounted-块站在低温恒温室内约1分钟或直到组织冷冻介质变得不透明。 </li><li>立即安装上述样品保持盘上朝上采样块的底侧的切片。使用刀片在样品块仍是相对"软"，以减少每个段的长度剪掉试样块的一部分（如果需要的话）。离开样品保持盘上的切片机至少5分钟，以允许它的温度达到平衡。 </li><li>渐渐地修剪样品块，直到蝌蚪的头是透过半透明明胶可见。以增加修剪速度，在此阶段应用部分厚度（ 例如 20-25微米）的高（厚）的设置（如启用"修剪"选项），但不能太厚，因为采样块可以从样品保持盘片脱落。观察低温恒温器的性能，如清晰度和叶片的角度在此阶段，以确保随后的部分的长条形的产生。 </li><li>一旦蝌蚪头变得可见，调节恒温器的设置恢复正常（ 如 10-12μM）和清洁都切片机和舞台用画笔。使用手轮设置（不使用机动设置）轻轻做2-3段作为试验，以确保成品片可以形成海峡IPS，不重叠或粘在叶片上。 </li><li>继续微调直到蝌蚪的头几乎暴露（这可能需要一些经验，但实现的）。刷过在舞台上的任何残余部分和样品部分的收集将开始。 </li><li>使大约10-15节，让他们形成一个长条形。 </li><li>厚厚的防护玻璃罩板翻转到一边，用细尖的油漆刷从刀片轻轻取出带，并安排其与平行于刀片长轴阶段。 </li><li>它迅速而坚定地接在室温下一个带正电荷的幻灯片，用铅笔（不会在随后的染色处理洗掉）贴上标签，按到具有朝下标签的一面带，并从低温恒温室的幻灯片。如果操作正确，条带应立即粘在带正电荷的滑动（ 图3A）。 </li><li>重复此步骤，对每个下滑20-30片e和并联布置（ 图3B）。这是足以覆盖X的整个大脑区域热带胚胎和X的最前脑和中脑区域蟾胚胎。 </li><li>风干载玻片10分钟，立即进行，或者在-80℃下，在滑动箱的幻灯片存储至少3-6个月而不丧失免疫原性。 </li></ol></li></ol> 3.分区域爪蟾胚胎的免疫染色<ol><li>准备实验试剂和材料。 <ol><li>通过加入的Triton X-100到1×TBS达到0.05％的终浓度制备0.05％的TBS-的Triton X-100。该溶液是在室温下3-4天是稳定的。不要使用过期的解决方案。 </li><li>制备5％热灭活的山羊血清或5％BSA的TBST作为封闭缓冲液。首先，通过在65℃水浴中放置约20-30毫升血清的30-60分钟热灭活的山羊/羔羊血清。分装成1.5毫升离心机管和在-20℃下冻结。无需重新灭活使用时需要。 </li><li>稀释的热灭活血清或BSA的TBST中使用之前达到5％的最终浓度。 </li><li> （:500抗SOX3 10,25 / 1:250抗MYT1 26 /抗乙酰微管蛋白如 1）根据在封闭缓冲液中稀释准备适当的初级抗体。使用约100微升抗体溶液每张幻灯片。 </li><li>在封闭缓冲液（500通常1种）制备适当的荧光抗体（ 例如抗小鼠/兔红荧光染料缀合的抗体）。 </li><li> （可选）添加红色荧光染料缀合的鬼笔环肽（1:500）到第二抗体混合以显示肌动蛋白网络。 </li><li> （可选）添加DAPI（0.5微克/毫升终浓度）纳入中学抗体混合物以可视化的核定。 </li><li>取抗褪色封固剂从冷冻的，并在室温水浴解冻。 </li></ol></li><li>免疫染色<ol><li>从-80℃中取出冷冻的幻灯片，并将其放置在通风罩内一张纸巾，为至少1小时，以消除任何冷凝水滴，再烤用一个85-90℃加热块干燥的幻灯片朝上15分钟的切片以激活粘附机制。最后，让该滑动冷却至室温（至少10分钟）。 </li><li>填用纯丙酮的染色缸，并培育在丙酮幻灯片10分钟，以除去鱼明胶。如果多个幻灯片将被处理，安排他们上的染色机架上。空气干燥处理，载玻片用于在通风橱15分钟。不要重复使用丙酮。 </li><li>认真借鉴使用PAP笔滑样品周围环不接触样品。确保环是自我封闭，否则抗体溶液染色会期间流出。充分干燥的PAP笔环。 </li><li>装满1X TBS另一染色缸不含Triton X-100。 Insert是干燥-滑入染色缸，并允许再水化至少1小时。在此期间，通过稀释或热灭活的山羊血清或BSA使用1×TBS用0.05％的Triton X-100 5％的最终浓度制备封闭缓冲液。 </li><li>通过将6月1日至2日孔板点击锁食品箱内做一个湿盒用超纯水中途补水井。从染色缸除去再水化载玻片和水平上的6孔平板放置（不板盖）。 </li><li>小心加入300-600微升的PAP环内封闭液。通过锁定盖就位密封湿盒，并在室温下孵育至少1小时。 </li><li>稀释适当初级抗体在封闭缓冲液。一般情况下，使用每张幻灯片100-150微升稀释抗体溶液。如果多张幻灯片进行处理，按比例扩大的体积。 </li><li>封闭后，小心地吸去除幻灯片封闭液快速添加primary抗体溶液，以防止干出，然后密封湿盒，并在4℃孵育过夜。 </li><li>第二天，移除载玻片通过抽吸第一抗体溶液。通过插入填充用1×TBS用0.05％的Triton X-100，3次，每次15分钟染色罐子洗幻灯片。同时，稀释适当荧光二级抗体与封闭缓冲液（具有或不具有DAPI或鬼笔环肽）。 </li><li>小心加入100-150微升的二级抗体溶液到幻灯片。将幻灯片湿箱内和密封盖子。孵育载玻片在室温下1-2小时。 </li><li>在充有1×TBS用0.05％的Triton X-100，3次，每次15分钟染色罐子洗幻灯片。在最后的洗涤中，如果储存在-20℃下解冻，在50℃水浴中的抗褪色封固剂10分钟。使用前冷却安装介质室温。 </li><li>添加约20微升（一滴）安装介质的上幻灯片和应用的大盖玻片（至少22毫米×64毫米）以上的样本。通过观察在6小时后安装荧光或共聚焦显微镜。 <ol><li>如果成像不能在同一天进行，使用指甲油密封幻灯片，并将其放置在湿盒内，4℃过夜。 注意:防褪色的安装介质将逐步得到几天后氧化（并打开褐色），因此，建议尽快拍摄的图像。 </li></ol></li></ol></li></ol>

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作者什么都没有透露。

这里，我们证明了在爪蟾胚胎可视化的主要神经发生了方便快捷的方法。此方法允许不同类型的神经细胞，包括使用细胞类型特异性标记物的神经元干细胞和分化的原代神经元的评估。 该协议是普遍强劲具有非常高的水平的再现性。对于那些在预孵化阶段的胚胎（ 即高达28期），我们建议您手动删除固定之前，卵黄膜，因为这可以让胚胎固定前充分舒展。在相对早期阶段收集时胚胎（孵化前）时，由于弯曲的胚胎是极难在安装室内部的期望的方向来安排，这是特别重要的。我们没有经历免疫原性MEMFA或PFA固定，因此附加抗原修复工序之后的损失是没有必要的此协议。此外，这种免疫染色过程可以在样品从显色/荧光原位杂交来进行。在这样的情况下，建议跳过原位杂交协议期间蛋白酶K处理步骤。色/荧光底物反应后，将样品可以用TBS洗涤并嵌入鱼明胶溶液中以类似的方式MEMFA / PFA固定的样品。样品瓶可以通过包裹在铝箔小瓶如果荧光原位杂交将连同免疫荧光染色后成像避光。 在一些情况下，胚胎可能明胶渗透后过度收缩。这是最有可能不是固定不充分或不够TBS-的Triton提取造成的。确保胚胎已被固定在PFA / MEMFA至少2-3小时或最好过夜4℃。如果问题仍然存在，加大TBS-的Triton的洗涤时间，以3 1小时。 在低温sectio宁，样本块的刚性可能会有所不同鱼明胶作为不同批次具有不同的属性。这个问题可以通过调整切片机的温度来部分地补偿。较低的温度会导致一个"难"样本块然而在过高的温度低样本块变得脆，难以部分。一些微调或每次练习之前切片（使用模拟样本块无胚胎）可能是有益的前上使用特别有价值的样品。 切片过程中经常遇到的问题是薄片有时往往粘在厚玻璃板上，而不是留平在钢阶段。这可能是特别破坏，因为它经常中断连续切片过程。节室和（特别是）厚玻璃板不是机器和o在够冷，或过多的静电荷:此类案件通常由两个原因之一perator，特别是在干燥的天气。前者可通过转动向下部腔室的温度和离开内的玻璃板额外的时间（可能是过夜）来解决;而后者通过适当的接地低温恒温器。低温恒温器的金属表面连接到一个金属抽头或金属制成的类似水管道系统可以以释放静电荷的另一种方式。每次使用后，在厚玻璃板应很好非腐蚀性洗涤剂（例如餐具洗涤液），通过超纯水漂洗洗涤，用纯乙醇喷洒，并包裹在毛巾纸保护表面和边从有害的。 在协议中列出的抗体通常是具体的，我们很少遇到的非特异性吸附或高背景信号。应当指出的是，如果用热灭活的山羊/羔羊血清作为封闭剂，过量的热失活（如可视化通过在血清中形成蓬松沉淀剂）应避免，因为这沉淀是到的第二抗体极具吸引力，并可能有助于高背景的来源。 这是可能的，有时需要，以执行双重染色同时形象化神经元细胞的不同群体。这种双重染色是可能的，一般给出了以下组合令人满意的效果:SOX3 / N-浴缸或MYT1 / N桶。然而，SOX3和MYT1的双重染色是由一个事实，即两种抗体都来自兔原点复杂。我们已经测试了几个抗体直接标记的试剂盒，但没有令人满意的结果，观察到（低信号对噪声电平），这可能是由于缺乏从二级抗体信号放大。一个可能的方法来解决这个问题将是提高在爪蟾的转基因株系，如下面所讨论。 其中一个此协议的主要限制是，虽然该协议能充分迪stinguish神经元细胞的两个主要的池，即SOX3表达​​神经干细胞池和MYT1表达分化的神经元，但它缺乏揭示分化的神经元的不同亚群的能力。这样的子种群，这包括，但不限于，主运动神经元，的interneurons，和感觉神经元，通常特征在于它们的差异表达的标记基因28-30。如上所述，最近在多色的原位杂交在爪蟾胚胎的基于抗体的免疫检测方法相结合的发展进步可以在间隙中填以显示分化的神经元的这些亚种群潜在调查31。另外，如在小鼠模型已被证实，它也将被期望的，以提高更全面组细胞类型特异性抗体的爪蟾区分这样不同的细胞群。 它是还值得一提的是，作为除了该方法，最近在光学成像和图像分析的进步，如多光子显微镜，三维重建，和分割，也可以应用于之后的初始评估，以实现爪蟾卵母细胞的更全面的观察以及早期胚胎，特别是在现场设置32,33。因此，要跟踪的增殖，分化，和在活体动物神经元细胞的运动，将它希望建立窝藏通过细胞类型特异性启动子驱动的荧光蛋白质，以允许在爪蟾早期这样的细胞群体的活观察中的一个或多个转基因品系胚胎。建立一个X的与特定的神经β微管蛋白启动子驱动tauGFP及其应用蟾线提供了一个很好的例子23,34。既SOX3和MYT1特征脊椎动物35-37的完整启动子序列，它是SH乌尔德相对容易地建立在爪蟾附加转基因品系应广泛地两个爪和初级神经发生研究的更一般的领域作出贡献。

在脊椎动物中，对中枢神经系统的发展包括几个独特的但连续的阶段。第一步是神经诱导，当幼稚外胚层细胞向神经命运，而不是表皮命运指定。几个相互连接的监管机制在这个阶段在非洲爪蟾和其他模型系统1,2参与。此过程主要由底层中胚层产生的分泌的因素，例如脊索头发生素，和卵泡抑素3-7协调。神经诱导之后，神经祖细胞的一个子集退出细胞周期，并开始在被称为主神经发生的方法来区分。不是所有的神经元前体分化在这个时候。剩余的神经前体细胞继续增殖，从而保持所需的整个发展和到成年中枢神经系统的持续增长的干细胞池。 这些公关oliferating神经前体细胞通过它们的SRY（性别决定区Y）-box 3（SOX3）基因8-11的表达为特征。细胞的其他人口，其出口处的细胞周期和承诺分化命运，由分化基因标记， 微管蛋白的表达，β确定2B IIb类 （tubb2b，N-桶 ）和髓鞘转录因子1（MYT1） 12-14。这种分化的神经元细胞最终产生不同类别的神经元，包括但不限于，电动机，间，和神经管15-17内定位在不同的区域的感觉神经元。 虽然显著努力，一直致力于揭露支配的图案和命运确定前神经外胚层事件的监管机制，较少注意已经取得了查处发生后的神经性事件初始阶段的图案。事实上，信号转导，转录法规，以及翻译后修饰都参与了这一后期，神经18-20期间同时控制定时和谱系规范。进一步调查这些机制需要一个可靠的方法来轻松查看并区分神经细胞的不同人群。上述神经标记物，包括，SOX3，MYT1，和N-浴盆，可用于识别这些不同的细胞群体，从而提供为揭示神经元分化21-23的基本机制的必要基础的装置。 虽然神经元细胞群体的差异化标记已经在其他模式生物已经证明，相对很少有研究利用了爪蟾系统在这方面充分。这主要是由于兼容的抗体的缺乏可靠地识别各种neurOnal地区的细胞群中的神经管。在这里，我们描述了通过免疫染色，它提供了在爪调查初级神经发生一个强大的和方便的方法在早期爪蟾胚胎可视化神经元分化的方法。这个协议应该给舞台26级45之间有意在非洲爪蟾中枢神经系统的早期开发的研究人员充分的指导。

<table><tbody><tr><td>gentamicin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>G1397</td><td></td></tr><tr><td>Tris-HCl&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T3253</td><td></td></tr><tr><td>Tris-base</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>NaCl&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>746436</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>746452</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>793639</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>MOPS</td><td>Sigma-Aldrich</td><td>RDD003</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>E3889</td><td></td></tr><tr><td>37% formaldehyde&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F8775</td><td></td></tr><tr><td>Gelatin from cold water fish skin</td><td>Sigma-Aldrich</td><td>G7765</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S7903</td><td></td></tr><tr><td>Base Mould Disposable 15 Mm x 15 Mm x 5 Mm Simport</td><td>VWR</td><td>EMB-200-015A</td><td></td></tr><tr><td>Base Mould Disposable 7 Mm x 7 Mm x 5 Mm Simport</td><td>VWR</td><td>EMB-200-010K</td><td></td></tr><tr><td>Superfrost Plus slides</td><td>ThermoFisher</td><td>12-550-15</td><td></td></tr><tr><td>Tissue Freezing Medium (OCT)</td><td>Leica&amp;nbsp;</td><td>14020108926</td><td></td></tr><tr><td>Cryostat</td><td>Leica&amp;nbsp;</td><td>CM3050</td><td></td></tr><tr><td>Gloves</td><td></td><td></td><td></td></tr><tr><td>Dry Ice</td><td></td><td></td><td></td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>&amp;nbsp;85-90 &amp;deg;C Heat block</td><td></td><td></td><td></td></tr><tr><td>PAP pen</td><td>Sigma-Aldrich</td><td>Z377821</td><td></td></tr><tr><td>Acetone, analytical grade</td><td></td><td></td><td></td></tr><tr><td>Staining jar with slide racks</td><td></td><td></td><td></td></tr><tr><td>Click-lock food box of appropriate size (has the capacity to hold 1-2 6-well plate inside)</td><td></td><td></td><td></td></tr><tr><td>anti-acetylated tubulin</td><td>Sigma-Aldrich</td><td>T7451</td><td></td></tr><tr><td>Anti-mouse IgG (H+L), F(ab&#039;)2&amp;nbsp;Fragment (Alexa Fluor&amp;reg;&amp;nbsp;488 Conjugate)</td><td>Cell Signaling</td><td>4408</td><td></td></tr><tr><td>Anti-rabbit IgG (H+L), F(ab&#039;)2&amp;nbsp;Fragment (Alexa Fluor&amp;reg;&amp;nbsp;555 Conjugate)</td><td>Cell Signaling</td><td>4413</td><td></td></tr><tr><td>Alexa Fluor&amp;reg; 647 Phalloidin</td><td>Cell Signaling</td><td>8940</td><td></td></tr><tr><td>DAPI</td><td>Cell Signaling</td><td>4083</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>Life Technologies</td><td>P36930</td><td></td></tr></tbody></table>

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.

代表性的结果显示在不同的级别，即前脑，中脑，后脑，和脊髓，用不同的抗体（ 图4）染色阶段30 爪蟾胚胎的横截面。如所提到的，SOX3标记神经元祖细胞群定位在神经管的内腔的附近，而MYT1标记的分化原代神经元向外迁移，并找到神经管的边际层（基底层）附近。抗乙酰微管蛋白标签在分化的神经元，其可在整个神经管内观察到轴突。 侧特异性基因敲除或过度表达是在神经生物学一个广泛使用的方法，以评估是否调节特定基因（多个）的表达破坏神经元细胞的生长和分化。在这样的情况下，无论是吗啉代（MOS）或DNA构建体携带亲<html> MOTER驱动基因（多个）在两细胞阶段23被注入到两卵裂球中的一个。可替代地，它们可以被注射到脑室，随后通过电穿孔，这将导致在击倒或过表达所需基因（多个）27的。在这两种情况下，其影响将被限制在胚胎的一侧，使得胚胎作为未处理的内部控制的相对侧。 固定，切片和免疫染色后，基因敲除/超表达的影响是通过计数和在胚胎的两侧比较不同种群的细胞数进行定量。通过累加从多个胚这些数据，可以进行统计分析。在我们的代表性的结果，没有扰动在胚胎中进行。基因扰动以及它们对不同的神经元细胞群的影响的实例可在参考文献20,23中找到。 T"FO:保together.within页="1"&gt; <img alt="图1" src="/files/ftp_upload/53949/53949fig1.jpg" /> 图1:胚胎排列和取向在部分模具 （A）的卡通描绘示出了安装组件，请注意托盘已经标有日期，阶段，胚胎的取向。 （b）显示安装组件的自然外观的图像。 <a href="https://www.jove.com/files/ftp_upload/53949/53949fig1large.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/53949/53949fig2.jpg" /> 图2:方向和取样保持光盘上的阿胶块的位置 （A）卡通描绘出的部分块组件，请注意，胚胎处于"抬起头"的位置和阿胶块牢固地固定Øn要通过在碱的组织冷冻介质中的样品保持盘（见图2B）。 （B）显示部分块组件的自然外观的图像，请注意阿胶块和取样保持盘之间的组织冷冻介质的累加。 <a href="https://www.jove.com/files/ftp_upload/53949/53949fig2large.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/53949/53949fig3.jpg" /> 图3:一个断面硐室连续切片 （A）型号。试样夹持盘应旋转，并且固定到该胚胎面朝上的位置。几（6-10）的部分后，连续部瓷砖的长条形被轻轻地从刀片（蓝色）用细刷分离，然后转90°的保持板。然后，一个房间温度带正电的幻灯片牢牢压在部分带朝下，马上举起收集到的成品带钢。 （B）的正常情况下，各滑动可以容纳条2平行线，如图所示。记住要使用铅笔幻灯片标签上详细记录。 <a href="https://www.jove.com/files/ftp_upload/53949/53949fig3large.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/53949/53949fig4.jpg" /> 图4:级30 X 的横截面 蟾 在神经组织蝌蚪和染色，20X。（A）的示意图表示剖面的上爪蟾蝌蚪的相对位置（前脑，中脑，后脑，和脊髓）。 （B）用抗SOX3（红色），抗乙酰化微管蛋白（格力染色对应的横截面n）和DAPI（蓝色），示出神经干细胞库，以及神经丝的神经管内的相对位置。 （C）的通讯用抗MYT1（红色）和DAPI（蓝色）染色的横截面，示出了有区别的原代神经元的神经管内的相对位置。比例尺= 20微米。 <a href="https://www.jove.com/files/ftp_upload/53949/53949fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining at JoVE.com

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

本文介绍了使用上的部分的免疫荧光染色爪蟾胚胎的中枢神经系统的可视化不同的神经元细胞群既方便又快速的方法。

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 ...

assessing-primary-neurogenesis-in-xenopus-embryos-using-immunostaining

Research

JoVE Journal

Neuroscience

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

9.5K 次观看.  University of Manchester. 本文介绍了使用上的部分的免疫荧光染色爪蟾胚胎的中枢神经系统的可视化不同的神经元细胞群既方便又快速的方法。 

视频: Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

在子宫内的电穿孔其次原发性神经文化为研究基因的功能在皮层神经元的一个子集

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

科研

神经科学

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

干细胞样<em&gt;爪</em&gt;胚胎外植体，研究早期神经发育特点<em&gt;体外</em&gt;和<em&gt;在体内</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

发育生物学

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

胚胎血管生成的可视化和定量分析<em&gt;热带非洲爪蟾</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

的神经植体培养<em&gt;非洲爪蟾</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

将DNA微注射到异种胚胎的眼蕾中，以及GFP表达光学轴状轴向轴向的成像，活的Xenopus大白鼠

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

荧光钙成像及其后续原位杂交，用于异种性前体特征

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

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

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

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 ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

从E14-E16斯普拉格·道利大鼠胚胎分离的混合原发希马和皮质神经元生成神经球

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

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/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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