我们想了很多精辟的讨论和B Novitch抗体的一种恩赐，感谢沙龙大话FOXP1。资助这项工作从威康信托基金会的助学金KCT和SRP（的格兰特参考号094399/B/10/Z）从威康信托基金会的项目资助。

1。制作所需的解决方案<ol><li> 1 M磷酸盐缓冲液:称取的109.4克Na 2 HPO 4和32克的NaH 2 PO 4 H 2 O中 ，混合和溶解在水中，至总体积为1升。 </li><li> 磷酸盐缓冲盐水（PBS）中:准备的0.1M磷酸盐缓冲液，0.15M NaCl的。 100毫升PBS中应足以从10胚胎的处理的片。 </li><li> 琼脂糖:热的PBS溶液至约70℃，并缓慢加入固体低熔点琼脂糖同时不断搅拌。使该溶液至终浓度为4％（重量/体积）的琼脂糖。使50毫升该溶液中的库存。发布此解决方案水浴中，保持在48℃，直到需要。实验后的未使用的任何的琼脂糖溶液在4℃下，可以存储为几个星期。 </li><li> 抗体块解决方法:添加小牛血清和Triton X-100的到最后concentr的梁漱溟的1％（V / V）胎牛血清，0.1％（体积/体积）的Triton X-100的PBS。 5片冰冻切片的免疫块解决方案的10毫升就足够了。 </li><li> 固色剂:准备0.75毫米氢氧化钠，4％（W / V）多聚甲醛（小心）。为了制备固定液热所需的量的水至70℃，添加浓NaOH到所需的最终浓度，添加固体多聚甲醛和混合直至溶解。冷却冰至4℃。 10毫升该溶液就足够了的20口油井的胚胎切片。 </li><li> 蔗糖溶液:准备10毫升含0.1M磷酸盐缓冲液的30％（重量/体积）蔗糖溶液。 10毫升该溶液就足够了胚胎切片和10口井。 </li><li> 70％（体积/体积）乙醇，70毫升无水乙醇稀释，用30毫升的水。 </li><li> 培养基:准备的溶液的1％鸡胚胎提取物，1％青霉素/链霉素，0.35％L-谷氨酰胺，0.1％2 - mercaptoethanol，4％鸡血清，2％B27补充剂和100纳克/毫升ciliaryneurotrophic的生长因子（CNTF）所有在neurobasal培养基稀释;制备在冰上，并用的同一天。 10毫升培养基所需的20井胚胎切片。 </li></ol> 2。鸡胚制备<ol><li>地点受精母鸡鸡蛋的最长边的水平在强制通风孵化器在38°C，直到他们开发阶段15 24。这通常需要大约4天的潜伏期。 </li><li>在培养的第二天，灭菌的蛋壳，浸泡在70％乙醇用纸巾擦拭。小心地刺进使用21号针头连接到一个5毫升注射器取出5毫升白蛋白的蛋壳的扁平端。这降低了胚胎离壳所以胚胎的外壳被打开时，不被损坏。返回蛋的孵化器和孵化他们的另外2天。 </li><li>用迟钝杜蒙特＃5镊子Çarefully捅破了约9厘米2的外壳顶部中间的外壳和删除。周围的胚胎切割，并小心的胚胎，并在HBSS剥离时。胚胎应根据汉堡和汉密尔顿（1992）15上演。所有的胚胎应该是在近阶段24。任何的胚胎表现出任何异常迹象，应被丢弃。 </li><li>删除羊膜，绒毛膜尿囊膜，头部和尿囊两个杜蒙特＃5镊子。剔骨的胚胎取出所有内脏器官。要做到这一点，剖开腹中的胚胎微解剖剪刀，一个钳子举行的胚胎周围的喙主干区域，抢头端的一部分，肠道和心脏和尾部拉。重要的是，这样做是干净的。你应该能够清楚地看到胚胎体节。一半，横向切胚胎之间的上肢和下肢buds.Now修剪胸体采用显微切割剪刀墙。 </li></ol> 3。胚胎切片的制备<ol><li>卸下的琼脂糖溶液从水浴中。一次性3毫升的塑胶巴斯德吸管的底部切5厘米。 </li><li>轻轻地取出腰椎的解剖胚胎用两个镊子摇篮的胚胎解剖的解决方案，并将其放置在一个干燥的培养皿中的一半。使用巴斯德移液管，取出约2毫升的琼脂糖，吸移管约0.5毫升的胚胎的顶部，然后进入移液管吸胚胎切片。在胚胎转移和琼脂糖进行剥离，远离塑料模具照顾，不引入任何气泡在琼脂糖。轻轻地将胚胎在塑料模具的底部的琼脂糖与胸部朝下。胚胎应定位要直接使用21号针头。切片也将包含在显影肢体的一部分，它是重要的，在琼脂的胚胎的取向OSE会允许这样做。将艇上冰的琼脂糖。要小心，胚胎不改变方向在此期间（约2分钟）。 </li><li>徕卡:VT1000S vibratome（速度，频率，400微米厚的片），应设置与切片室充满了HBSS，周围环绕着冰冷的水。琼脂糖块，然后粘超级胶水的vibratome平台。在琼脂糖块修整用剃刀刀片与它周围的琼脂糖1-3毫米离开胚片。选择，包括肢芽的部分有明显的顶外胚层rigde切片，这些切片，然后放置在HBSS。一般来说，有只有一到两个合适的片每个胚胎。轻轻地取出琼脂糖周围的组织切片，用两针。至关重要的是，这样做是彻底和仔细。 </li></ol> 4。培养胚胎切片<ol><li>加入500毫升培养液的必需参数（如上所述）24孔超低附件处理的组织培养板的孔的数目。片小心地从入井的HBSS溶液转移。在一般情况下，我们尝试在各孔中有两到三个切片。 24孔组织培养板在培养箱中在37℃下放置24小时，用5％的CO 2。 </li></ol> 5。切片的切片，免疫组化染色<ol><li>文化时期，加入500μl的无缓冲修复，每口井的胚胎切片，留在冰上20分钟。总溶液然后小心地除去和3的PBS洗涤，用5分钟的时间间隔进行。的切片，然后留在蔗糖溶液，在4℃，直到切片水槽中，约6小时，但可以离开的时间（ 例如过夜）。 </li><li>约5分钟，取出切片，然后轻拍掉额外的蔗糖溶液中平衡约1毫升OCT在20°C每片安装在一个充满OCT-剥离的塑料模具持平。安装后，将模具垂直上干冰巩固。 </li><li>安装OCT块，在-24°C的低温恒温器和平衡块约20分钟。将每个切片，用15毫米的部分安装对的Superfrost加幻灯片。幻灯片可以储存于-80℃直到需要它们。 </li></ol> 6。免疫<ol><li>移液器1-2毫升PBS持在潮湿的腔室与从腔室底部（我们使用1或2毫升塑料移液器用高压釜磁带固定）提出的幻灯片上水平滑动。孵育在PBS中的部分在20℃下5分钟，以除去过量的OCT取出PBS溶液从滑动和块溶液加入500毫升的部分，温育30分钟，在20℃下删除的块的解决方案，并立即加入500毫升稀释的抗体添加到幻灯片，在4°C孵育18-22小时。 </li><li>移除第一抗体溶液，并立即h的幻灯片用1ml PBS中5分钟，3次，在20℃，以除去过量的第一抗体溶液。加入500毫升稀释后的二抗的部分。在20°C 30分钟的幻灯片，然后离开。的二次抗体溶液，然后除去和滑动洗涤3次，在20℃下在PBS中的5分钟</li><li>这些清洗后，取出最后PBS冲洗，DAB的幻灯片边缘以去除多余的PBS，加两滴vectashield幻灯片上的，轻轻打下了的盖玻片超过的部分，避免泡沫的形成。删除多余的Vectashield印迹从之前的幻灯片的边缘成像。 </li></ol> 7。成像<ol><li>图像的免疫染色的部分。我们使用了尼康的Eclipse E80i配备了一个Hamammatsu ORCA ER数码相机，10倍，20倍和40 X物镜的荧光显微镜。 </li></ol>

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的作者都没有相互竞争的利益或利益冲突。

这里所描述的协议允许的鸡胚胎切片保温一天以上，同时保持运动神经元的存活，并继续培养期间迁移。在阐明的条件，以保持运动神经元的存活，我们已经确定了几个主要特点。高达4％的小鸡血清它似乎是关键的运动神经元的生存和它的替换与来自不同物种的血清是不能容忍的。有趣的是，我们发现，较高浓度的血清的存在下，不利的，因为它们促进导致严重扭曲切片中的形状的组织过度生长的切片。神经营养因子cilliary的存在下，更重要的是，也被证明是至关重要的。它仍然是一个明显的可能性，可能可以相当长的时间，比只是24小时进行培养，甚至允许可视化感觉传入输入到脊髓切片。此外，CULTURE条件，也可能是有用的切片文化的发展脑干和中脑/小脑。 或许这些切片的培养条件下，在使用的最大的潜在的好处是，它们促进蛋白质的功能，同时尽量减少潜在的胚胎的其余部分上的不利影响，如心脏药理操纵的可能性。大量的不同的信号通路抑制剂和激动剂可用于评估不同的脊髓神经元亚型的迁移和潜在的，他们在开发过程中形成的局部脊髓电路的影响。此外，一些基因蛋白的表达，如那些利用siRNA和吗啉代寡核苷酸，受到扰动的事实，他们只能在开发初期推出（中在OVO电过程碍于）和效果只持续短期内添E（通常为一天）。我们的片文化在稍后阶段开始，并让使用这种技术的可能性在发展期间，文化的切片切片阶段，以查看效果。 片培养协议，也可以让双方脊髓运动神经元，以及作为背interneuron的迁移时间的推移实时通过视频显微镜的可视化。这可以实现白色光下使用相差显微镜或通过使用荧光成像。如果后者的技术被使用，则需要引入荧光标记。这可能通过引入如荧光染料DiI荧光或DIO来实现，也可以通过任逆行标记从肢体或顺行标签从心室或卵内的电驱动荧光蛋白，在神经元的一个子集的结构。

在正常胚胎发育过程中，许多不同类型的细胞生成，必须从他们的角度原产地迁移，在那里他们将最终在成熟的有机体的功能。脊髓内的显影，在中线位于脑室区祖细胞，并生成许多亚型的有丝分裂后的神经元和神经胶质细胞，这些细胞必须迁移到融入功能的神经元的感官处理所需的电路和电机输出1。脊髓运动神经元控制四肢肌肉的收缩已被广泛研究，多是已知的分子和机制，推动形成不同的子类。然而，更何况是运动神经元迁移，分离和接收突触输入的机制。 运动神经元亚型的身份获得在开发过程中在一个分层的方式。运动神经元亚型可大致归我置身于不同的列，被定义为他们的轴突预测的部门和游泳池。汽车列项目轴突为轴向，内脏或四肢的肌肉。汽车部门细分的肢体投影到那些投射到腹部或背部肌肉的运动神经元电机外侧柱（LMC）。运动神经元群内的部门，被称为电机池项目单独的肌肉，肢体的2，3。肢体突出的运动神经元中定义的由它们的表达的转录因子FOXP1 4，5，而分割的身份是其特征在于，表达的同源结构域的转录因子胰岛-1（腹侧凸）或LHX-1（背侧凸）6。事实上，LHX-1的表达所需的适当的背突起的运动神经元7。这些亚型占据不同的位置，在脊髓腹侧突出的运动神经元来居住内侧背投影法纳克运动神经元。结果被隔离在大麦哲伦星云所谓的LMCmedial（LMCm）和LMClateral（LMCl）部门。池组织部门和电机的收购分为两个阶段8。在第一阶段中，分割偏析实现通过侧向时尚内侧的特性的运动神经元的迁移。 LMCl细胞后，将产生的LMCm细胞等LMCl的通过的LMCm移动实现其最终停留位置。第二阶段运动神经元内的分工和集群成运动神经元池，大概是通过一个背腹排序，运动神经元。已经示出连环蛋白依赖的古典钙粘蛋白家族的成员，是至关重要的两个阶段的运动神经元组织8-10。然而，钙粘蛋白功能如何控制细胞运动知之甚少。 收购柱状，部门和池标识需要外在的信号，这种模式本质安全C表达的转录因子11。此外，约50％的运动神经元死亡通过细胞凋亡在正常发展的一个过程，需要从肢体的外在信号，主要是通过神经营养支持的运动神经元生存。因此，运动神经元的发展，需要适当的环境，以保持在一个相对长的时间过程。出于这个原因，产生脊髓切片培养的实用性，以保持运动神经元存活所需要的适当的培养条件下的澄清。往前的胚胎切片培养已被用于跟踪的行为非本质加入纯化的神经元群体的12-14。然而，据我们所知，在片本身在原地运动神经元的存活和迁移的培养条件，促进尚未公布。我们修改一个标准的文化条件，有利于生存的纯化，分离颅运动神经元，以方便米OTOR神经元内的胚胎切片文化系统的发展。我们还监测了定位尚存的运动神经元的，并表明主要为正常的位置的运动神经元师培养期间获取。

<table border="1"><tbody><tr><td>鸡蛋</td><td>英国亨利斯图亚特农场， </td><td></td><td> Fertilised布朗Bovan金母鸡的蛋（亨利·斯图尔特农场，英国），保存在14°C，直到孵化</td></tr><tr><td> 21号针</td><td> BD-Microlance 3 </td><td> 304432 </td><td></td></tr><tr><td> 5毫升注射器</td><td> BD Plastipak公司</td><td> 302187 </td><td></td></tr><tr><td> ＃5杜蒙钳</td><td> Roboz </td><td> RS 5045 </td><td></td></tr><tr><td>微解剖剪</td><td> Roboz </td><td> RS 5910SC </td><td> （3.5中直） </td></tr><tr><td>胚胎嵌入模具:剥离模具</td><td>琼脂科学</td><td> G3764 </td><td>金字塔形22毫米至12毫米</td></tr><tr><td>超能胶</td><td></td><td></td><td>电源的Flex，乐泰</td></tr><tr><td>乙醇</td><td> SIGMA </td><td> 32221 </TD&gt; <td></td></tr><tr><td>钠，二氢磷酸盐，单水合物</td><td> BDH </td><td> 102454R </td><td></td></tr><tr><td>迪单钠，磷酸氢</td><td> VWR / BDH </td><td> 102494C </td><td></td></tr><tr><td>氯化钠</td><td> VWR / BDH </td><td> 27810.295 </td><td></td></tr><tr><td>低熔点琼脂糖</td><td> Invitrogen公司</td><td> 16520 </td><td></td></tr><tr><td>胎牛血清</td><td> Gibco公司</td><td> 10500-064 </td><td></td></tr><tr><td>的Triton X-100的</td><td> SIGMA </td><td> T8787 </td><td></td></tr><tr><td>氢氧化钠</td><td> FLUKA </td><td> 71689 </td><td></td></tr><tr><td>多聚甲醛</td><td> VWR / BDH </td><td> 28794.295 </td><td></td></tr><tr><td>一次性塑料巴氏吸管</td><td> ELKAY精密实验室消耗品</td><td> 127-P503-000 </td><td> Liquipette 3毫升的量</td></tr><tr><td> Hanks平衡盐溶液（HBSS）中</td><td> Gibco公司</td><td> 14170 </td><td></td></tr><tr><td>蔗糖</td><td> SIGMA </td><td> S0389 </td><td></td></tr><tr><td>碳酸氢钠</td><td> SIGMA </td><td> S5761 </td><td></td></tr><tr><td>鸡胚提取物</td><td> Seralabs </td><td> CE650-J </td><td></td></tr><tr><td>青霉素/链霉素溶液</td><td> Gibco公司</td><td> 15070 </td><td></td></tr><tr><td> L-谷氨酰胺溶液</td><td> Gibco公司</td><td> 25030 </td><td></td></tr><tr><td> 2 - 巯基乙醇</td><td> Gibco公司</td><td> 21985 </td><td></td></tr><tr><td>马血清</td><td>南方集团实验室</td><td> 9028B </td><td></td></tr><tr><td> B27的补充</td><td> Invitrogen公司</td><td> 17504-044 &lt;/ TD&gt; <td></td></tr><tr><td> Neurobasal培养基</td><td> Gibco公司</td><td>猫〜2103 </td><td></td></tr><tr><td> CilliaryNeurotrophic因子（CNTF） </td><td> R和D系统</td><td> 557-NT-010 </td><td></td></tr><tr><td>鸡血清</td><td> SIGMA </td><td> C5405 </td><td></td></tr><tr><td>一抗</td><td></td><td></td><td>购自发展研究杂交银行，除反FOXP1抗体，这是一种从Bennett Novitch加州大学洛杉矶分校的礼物。初级抗体稀释兔抗-FOXP1（1/32000），小鼠的抗-LHX1 / 2（4F2）（1/50），豚鼠的抗FOXP1（1/1000）。 所有的抗体在4℃下存储</td></tr><tr><td>第二抗体</td><td></td><td></td><td>杰克逊Immunoreseach Laboratories公司的驴抗小鼠（的Alexa Fluor 488）（1:1000），反主动购买的驴抗，豚鼠（CY3）（1:1000）EY（抗兔的Alexa Fluor 594）购自Invitrogen（1:1000） 所有的抗体在4℃下存储</td></tr><tr><td> 24 - 孔板</td><td>康宁COSTAR </td><td> 3473 </td><td>超低附件24 - 孔板</td></tr><tr><td> OCT </td><td>组织康，樱花</td><td> 4583 </td><td>最佳的切削温度</td></tr><tr><td> Vectashield </td><td>矢量实验室</td><td> H1000 </td><td> Vectashield荧光安装介质</td></tr><tr><td>幻灯片</td><td> Thermo Scientific的</td><td></td><td>的Superfrost的带正电荷的幻灯片，25×75×1.0毫米</td></tr><tr><td>盖玻片</td><td> VWR国际</td><td> 631-0167 </td><td> 25x60毫米</td></tr><tr><td>强制通风孵化器: </td><td>里昂电气公司</td><td></td><td>保持湿润​​，确保总是有足够的水位，保持在38°C </td></tr><tr><td>低温恒温器</td><td>布里特，亨廷顿，英国</td><td></td><td> -36°C </td></tr><tr><td>组织培养箱</td><td> DH自动流</td><td></td><td> nuaire 38℃，5％CO 2的 </td></tr><tr><td> Vibratome </td><td>徕卡</td><td></td><td>徕卡VT1000-S </td></tr><tr><td>显微镜</td><td>尼康</td><td></td><td>尼康的Eclipse的E80i荧光显微镜</td></tr><tr><td>数码相机</td><td>尼康</td><td></td><td>尼康DS5M和Hamammatsu ORCA ER </td></tr></tbody></table>

chicken embryo spinal cord slice culture protocol

切片培养的胚胎发育可以方便的操作都通过药理学和基因操作。这降低了系统潜在的致命的副作用，由于全身药物的应用是可以克服的。然而，文化条件必须确保正常发展所得的片内减少环境。我们一直专注于脊髓的发展，尤其是脊髓运动神经元。我们系统地不同的培养条件的鸡胚胎切片，的点最脊髓运动神经元诞生了。我们测定存活培养期间和运动神经元的位置的那些相比， 在体内的运动神经元的数目和类型。我们发现，培养期间需要血清型和神经营养因子，并能够保持为至少24小时，并允许这些运动神经元迁移到适当的位置中的运动神经元存活脊髓。我们提出这些培养条件和方法，准备使用嵌入在琼脂糖全净膛鸡胚胎的胚胎切片文化和切片用一个vibratome。

使用培养的片一直遵循的发展interneuron和投射神经元影响力的进展我们对组织产生的内皮层16。在脊髓运动神经元的发展一直遵循文化的整个斑马鱼胚胎17。然而，在斑马鱼的脊髓运动神经元组织是相对简单的（由于缺乏鱼的大型四肢肌肉）。该驱动器的脊髓运动神经元组织层次的高等脊椎动物的发展过程中的分子机制知之甚少。在高等脊椎动物的胚胎切片文化，促进神经元的存活和细胞体迁移，因此需要培养条件。目前，脊髓切片培养的高等脊椎动物已被用于种子解离的神经元的顶部片12-14，调查荧光标记的切片的外周中的神经轴突18，或祖细胞早期脊髓发育行为19。片培养条件后脊髓，尤其是那些保持运动神经元发展目前所缺乏的。 为了试图脊髓运动神经元脊髓神经元的发育，特别是，我们研究了各种文化条件，可能会促进运动神经元的存活和运动组织通过横向迁移的神经元的初始订单收购。使用阶段18阶段20鸡胚脊髓切片的初步试验证明无果而终，我们不能够保持运动神经元存活超过几个小时的时间，并不能证明代相当数量的LMCl细胞的培养周期（数据未示出）。因此，我们研究阶段，24胚片文化的一个时间点时，大多数的LMCl和LMCm神经元已经产生，但是当LMCl神经元的迁移仍然是在我nfancy（ 图1A-C）。这种横向的LMCl神经元的迁移，主要是完成阶段27，约24至36小时后（ 图1 DF）。因此，我们的分析可以被简化为能够保持活着的神经元，以协助他们迁移到腹角横向。我们开始我们的实验中，使用相对简单的培养条件下，只包含Hank氏平衡盐溶液的鸡血清或鸡胚提取物带或不带。在所有情况下，尽管我们能够保持LMC切片中的神经元，我们发现几乎没有证据表明LMCl神经元保持（至少LHX-1的表达）。此外，我们发现不适当表达的基因，如HB9在脊髓背角， 在体内的 （数据未示出）的情况下，永远不会发生。因此，这些简单的文化条件不适合的收购运动神经元组织的调查。 因此，我们找到了更复杂的条件[NS和使用的碱的配方已发布，以方便分离的鸡胚脑神经运动神经元的存活。此条件20（1％鸡胚提取物，1％笔链球菌，0.35％L-谷氨酰胺，0.1％2 -巯基乙醇，2％马血清，2％B27补充剂和50纳克/毫升ciliaryneurotrophic的生长因子（CNTF）在neurobasal培养基（这里称为格思里中等），并保持活着比更简单的媒体更多的运动神经元。此外，它没有导致在杂散表达的转录因子在脊髓背角。然而，在生存的LMCl神经元是穷人（ 图1 GI， p）的，因此，我们改变我们认为是关键因素的Guthrie介质，即动物血清的来源，血清中的浓度和睫状神经营养因子在培养基中的浓度，我们发现，改变从马血清的血清小鸡血清和CNTF的浓度增加，从50毫微克/毫升至100毫微克/毫升的大幅增加吨他的的LMCl和LMCm细胞的生存，也允许他们迁移到脊髓前角超过24小时的培养条件（ 图1 JL，P）。运动神经元的总数，比LMCm LMCl和，的确，迁移的LMCl细胞到腹侧角出现非常相似的部分的胚胎被允许开发卵内 ，而不是在该切片培养（ 图1 KO，P）。因此，我们认为，基于转录因子的表达，细胞数量和位置的运动神经元，切片培养条件概括正常的脊髓运动神经元的发展至少在24小时内。 <img alt="图1" fo:content-width="2in" fo:src="/files/ftp_upload/50295/50295fig1highres.jpg" src="/files/ftp_upload/50295/50295fig1.jpg" /> 图1。 A - C。代LMC的状态（FOXP1染色b）和LMCl（LHX-1染色在 a）中，在阶段24。 c是一个合并的两个通道。中线作为a和D中的虚线所示，V表示的脊髓背腹轴（D，V）的取向。D - F。 。G - 我的状况:LMC（d） 中 FOXP1染色和LMCl（LHX-1染色在 e）组织阶段27。f是一个合并的两个通道。 LHX-1（g）和FOXP1（ 小时 ）在部分支持颅运动神经元细胞的存活（我们的"初始培养基"的Guthrie介质，见参考文献20），在培养基中培养的切片中表达。 LHX-1的是，不再在运动神经元中表达，虽然有一些LMC证明FOXP1表达细胞的生存。 ，D，V（g）中示出的脊髓背腹轴（D，V）的取向。 ML显示的正中侧面（M，L）的s轴的方向皮纳尔线的J - 升 。 LMCl细胞（LHX在腹侧角在 j-1）和LMC一般（FOXP1 在 k）中可以观察到在含有4％鸡血清和100 ng / ml的睫状神经营养因子的培养基中培养的切片。请注意，在腹侧角的横向位置被发现在一些LMCl细胞。 （j）条中的箭头示出了一些的LMCl细胞的横向位置。 （L）是一个合并的两个通道。 米- O。 LHX-1（m）和FOXP1（n）中的胚胎内的胚胎在 j - 升所示的切片培养过程中保持在卵的表达状况。 （O）是一个合并的两个通道。定量的百分比的LMCl（FOXP1 + VE / LHX1 +）细胞与LMC（FOXP1 + VE / LHX1-VE）在片文化在不同条件下的细胞中发现的Çompared控制卵 （占100％） 中保持的胚胎。学生的t检验***表示p &lt;0.01。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50295/50295fig1large.jpg" target="_blank">点击此处查看大图</a> 。

Watch this Scientific Journal Video about Chicken Embryo Spinal Cord Slice Culture Protocol at JoVE.com

Chicken Embryo Spinal Cord Slice Culture Protocol

切片文化促进胚胎发育的基因和药理学的扰动操作。然而，文化条件必须确保可以继续正常发展的片内减少环境。我们说明了一个协议，有利于脊髓发育正常进行至少24小时。

鸡胚胎脊髓片培养协议

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

chicken-embryo-spinal-cord-slice-culture-protocol

Nanyang Technological University

Research

JoVE Journal

Neuroscience

22.0K 次观看.  University College London. 切片文化促进胚胎发育的基因和药理学的扰动操作。然而，文化条件必须确保可以继续正常发展的片内减少环境。我们说明了一个协议，有利于脊髓发育正常进行至少24小时。

视频: Chicken Embryo Spinal Cord Slice Culture Protocol

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

器官型切片文化绿色荧光蛋白表达的实时成像周围神经生长的小鼠胚胎

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

科研

神经科学

Preparation and Culture of Chicken Auditory Brainstem Slices

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at discreet periods of development 1-4 as well as mechanisms for temporal coding in the central nervous system 5-7.
Here we present a method to prepare chicken auditory brainstem slices that can be used for acute experimental procedures or to culture organotypic slices for long-term experimental manipulations.
The chicken auditory brainstem is composed of nucleus angularis, magnocellularis, laminaris and superior olive. These nuclei are responsible for binaural sound processing and single coronal slice preparations preserve the entire circuitry. Ultimately, organotypic slice cultures can provide the opportunity to manipulate several developmental parameters such as synaptic activity, expression of pre and postsynaptic components, expression of aspects controlling excitability and differential gene expression
This approach can be used to broaden general knowledge about neural circuit development, refinement and maturation.

The chicken auditory brainstem is comprised of nuclei responsible for binaural sound processing. A single coronal slice preparation maintains the entire circuitry while the cultured approach provides a unique preparation to study the development of neuronal structure and auditory function at the molecular, cellular and network levels.

鸡脑干切片的制备及文化

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

生物学

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are located in the dorsal spinal cord and send their axons along stereotyped trajectories. Commissural axons initially project ventrally towards and then across the floorplate. After crossing the midline, these axons make a sharp rostral turn and project longitudinally towards the brain. Each of these steps is regulated by the coordinated activities of attractive and repulsive guidance cues. The correct interpretation of these cues is crucial to the guidance of axons along their demarcated pathway. Thus, the physiological contribution of a particular molecule to commissural axon guidance is ideally investigated in the context of the living embryo. Accordingly, gene knockdown in vivo must be precisely controlled in order to carefully distinguish axon guidance activities of genes that may play multiple roles during development.
Here, we describe a method to knockdown gene expression in the chicken neural tube in a cell type-specific, traceable manner. We use novel plasmid vectors3 harboring cell type-specific promoters/enhancers that drive the expression of a fluorescent protein marker, followed directly by a miR30-RNAi transcript4 (located within the 3'-UTR of the cDNA encoding the fluorescent protein) (Figure 1). When electroporated into the developing neural tube, these vectors elicit efficient downregulation of gene expression and express bright fluorescent marker proteins to enable direct tracing of the cells experiencing knockdown3. Mixing different RNAi vectors prior to electroporation allows the simultaneous knockdown of two or more genes in independent regions of the spinal cord. This permits complex cellular and molecular interactions to be examined during development, in a manner that is fast, simple, precise and inexpensive. In combination with DiI tracing of commissural axon trajectories in open-book preparations5, this method is a useful tool for in vivo studies of the cellular and molecular mechanisms of commissural axon growth and guidance. In principle, any promoter/enhancer could be used, potentially making the technique more widely applicable for in vivo studies of gene function during development6.
This video first demonstrates how to handle and window eggs, the injection of DNA plasmids into the neural tube and the electroporation procedure. To investigate commissural axon guidance, the spinal cord is removed from the embryo as an open-book preparation, fixed, and injected with DiI to enable axon pathways to be traced. The spinal cord is mounted between coverslips and visualized using confocal microscopy.

In ovo Electroporation of miRNA-based Plasmids in the Developing Neural Tube and Assessment of Phenotypes by DiI Injection in Open-book Preparations

A method by which gene expression in the neural tube can be downregulated in a cell type-specific, traceable manner is described. We demonstrate how in ovo electroporation of microRNA-based plasmids that elicit spatiotemporally controlled RNA interference can be used to investigate commissural axon guidance in the developing neural tube.

基于miRNA的质粒在发展中的神经管和表型的评估DiI荧光注射开卷准备在卵的电

Commissural dI1 neurons have been extensively studied to elucidate the mechanisms underlying axon guidance during development1,2. These neurons are ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

脊髓培养法研究胚胎鸡组织的发展

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

发育生物学

Isolation and Culture of Dissociated Sensory Neurons From Chick Embryos

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. Studying neurons in vivo can be challenging due to their complexity, their varied and dynamic environments, and technical limitations. For these reasons, studying neurons in vitro can prove beneficial to unravel the complex mysteries of neurons. The well-defined nature of cell culture models provides detailed control over environmental conditions and variables. Here we describe how to isolate, dissociate, and culture primary neurons from chick embryos. This technique is rapid, inexpensive, and generates robustly growing sensory neurons. The procedure consistently produces cultures that are highly enriched for neurons and has very few non-neuronal cells (less than 5%). Primary neurons do not adhere well to untreated glass or tissue culture plastic, therefore detailed procedures to create two distinct, well-defined laminin-containing substrata for neuronal plating are described. Cultured neurons are highly amenable to multiple cellular and molecular techniques, including co-immunoprecipitation, live cell imagining, RNAi, and immunocytochemistry. Procedures for double immunocytochemistry on these cultured neurons have been optimized and described here.

Cell culture models provide detailed control over environmental conditions and thus provide a powerful platform to elucidate numerous aspects of neuronal cell biology. We describe a rapid, inexpensive, and reliable method to isolate, dissociate, and culture sensory neurons from chick embryos. Details of substrata preparation and immunocytochemistry are also provided.

分离和游离感觉神经元的文化活动从鸡胚

Neurons are multifaceted cells that carry information essential for a variety of functions including sensation, motor movement, learning, and memory. ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

人类干细胞异种移植到鸡胚

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

调查器官脊髓联合培养物中生长多电极阵列功能再生

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Slicing the Embryonic Chicken Auditory Brainstem to Evaluate Tonotopic Gradients and Microcircuits

The chicken embryo is a widely accepted animal model to study the auditory brainstem, composed of highly specialized microcircuitry and neuronal topology differentially oriented along a tonotopic (i.e., frequency) axis. The tonotopic axis permits the segregated encoding of high-frequency sounds in the rostral-medial plane and low-frequency encoding in caudo-lateral regions. Traditionally, coronal brainstem slices of embryonic tissue permit the study of relative individual iso-frequency lamina. Although sufficient to investigate anatomical and physiological questions pertaining to individual iso-frequency regions, the study of tonotopic variation and its development across larger auditory brainstem areas is somewhat limited. This protocol reports brainstem slicing techniques from chicken embryos that encompass larger gradients of frequency regions in the lower auditory brainstem. The utilization of different slicing methods for chicken auditory brainstem tissue permits electrophysiological and anatomical experiments within one brainstem slice, where larger gradients of tonotopic properties and developmental trajectories are better preserved than coronal sections. Multiple slicing techniques allow for improved investigation of the diverse anatomical, biophysical, and tonotopic properties of auditory brainstem microcircuits.

Here we present a protocol for obtaining non-coronal auditory brainstem slices of the chicken embryo for the investigation of tonotopic properties and developmental trajectories within one brainstem slice. These slices include sagittal, horizontal, and horizontal/transverse sections encompassing larger tonotopic regions within an individual slice plane that the traditional coronal sections.

对胚胎鸡听觉脑干进行切片以评估异位梯度和微电路

The chicken embryo is a widely accepted animal model to study the auditory brainstem, composed of highly specialized microcircuitry and neuronal topology ...