我们亲切地感谢约翰·海斯博士脚本，博士。埃里克·布拉德利和克里斯托弗·德尔 - 内格罗共聚焦显微镜的帮助;德鲁·休斯，劳拉ODORIZZI，亚历Garafalo，丽贝卡Lowden，和Liz麦克默里对他们的工作在发展该项目，并提供初步的数据统计分析有用的建议;格雷戈·史密斯博士。这项工作是由美国国立卫生研究院授予（NINDS IR15N5067566 01）MSS和霍华德休斯医学研究所的科学教育补助金的威廉和玛丽学院支持。

所有的实验都完成后的实验动物护理和使用委员会在威廉和玛丽学院批准的协议。在本协议中引用的发展阶段根据Nieuwkoop和Faber 46的 。 1。解剖<ol><li>允许的细胞培养液中的钙，镁免费中等（CMF），以平衡至室温。您还需要0.1X马克的改良林格（MMR）的pH值7.4-7.6。 </li><li>消毒通过施加在细胞培养层流罩30分钟的UV光对以下项目。 （喷每个资料前放置在通风橱中，用70％的乙醇）。 <ul><li> 35 mm塑料培养皿中。 </li><li> 60 mm塑料培养皿中。 </li><li> 35毫米Nunclon表面菜肴。 </li><li> 1.5 ml微量离心管。 </li><li> P1000微量移液器（1000微升微量移液器）。 </li><li>气溶胶抗P1000秘诀（1000微升微量移液器的气溶胶耐提示）。 </li><li>酒精耐笔。 </li><li>精细解剖钳。 </li><li> * 15毫升锥形管（仅用于成像活性钙）。 </li><li> *仅适用于活性钙​​成像。 </li></ul></li><li>一旦引擎盖已紫外线灭菌，解决方案具有平衡到室温，打开在通风橱中的空气层流，在通风橱中，用70％乙醇和地点媒体瓶喷洒下来手套和媒体瓶。 </li><li>油烟机内准备好以下P1000微量移液器与每块板的细胞 ，以确保适当的标签。 <ul><li>一个60毫米的塑料培养皿中含有10毫升的细胞培养基- ​​夹层板 。 </li><li>一台35毫米Nunclon面菜含有2毫升细胞培养基- ​​培养板 。 （Nunclon塑料盘子不进一步处理任何粘接剂）。 </li><li>一个空的1.5 ml离心管外植体解离管 。 </li><li> *一个15毫升锥形猎鹰管内含有15 ml细胞培养液中。对于冲洗掉多余的Fluo4-AM成像时，活性钙。 </li></ul></li><li>油烟机内，需准备以下与P1000微量移液器每个解剖届 （一个以上的视网膜可以在一个会话中剥离）。 <ul><li>一个35毫米塑料培养皿中含有2毫升CMF - 植冲洗板 。 </li><li>一个35毫米的塑料培养皿中含2毫升CMF - 外植体解离板 。 </li></ul></li><li>外罩准备以下内容： <ul><li>一个60毫米塑料培养皿中的细胞中含有10 ml的0.1倍MMR每板- 固定板 。 </li><li>一个60毫米塑料培养皿中的细胞中含有10毫升0.1X MMR每板- 兄弟姐妹板 。 </li><li>一个60毫米的塑料培养皿中含10毫升0.1X MMR + 0.5毫克/毫升MS-222（三卡因） - 麻醉板每清扫会话。 </li></ul></li><li加入40微升5％胰蛋白酶CMF外植体解离板 （0.1％的胰蛋白酶溶液），摇匀，并预留。 </li><li>如果解剖是年龄小于30期的胚胎，添加10毫克胶原酶B到夹层板 （在1 mg / mL胶原酶溶液B），漩涡溶解，并预留。 </li><li>选择所需阶段的胚胎，用非无菌移液管转移到控股板 ，并删除（如果尚未孵化胚胎卵黄膜）用细钳子。 </li><li>传输数个相同的上演胚胎到同级板与非无菌移液管。 </li><li>如果胚胎是25或更早的阶段，继续进行直接与夹层，否则的胚胎转移到的异丙酚板与非无菌移液管和允许胚胎坐下，直到停止运动，对刺激的反应。这可能需要长达一分钟。 </li><li>在解剖范围（目标4X，10X目镜）进行解剖（ 图2）。 </li></ol>胚胎阶段，25岁或以下 <ol start="13"><li>与非无菌移液管转移胚胎解剖板 。 </li><li>使用细钳子2双，作出正中矢状切腹侧的胚胎，在胚胎的前部的后端上开始，并继续通过水泥腺。 </li><li>小心地抓住任何一个边缘的剪裁和传播左，右打开的胚胎。这将导致在胚胎面临的夹层板的背侧，腹侧外胚层揭示的内胚层，中胚层和内流传开。 </li><li>开始除去内胚层，中胚层（ 图2A和2B）。这个组织最早，规模最大的层是在外观上与“蓬松”大细胞和小明显的组织。 </li><li>除去这层后，体节，脊索将变得可见。对于更好的对比度，将一个单独的60毫米的塑料培养皿中含有10毫升细胞培养基和100μl耐尔蓝硫酸盐1％溶液（在水中）的回传的夹层板之前的2-3分钟的胚胎。这会弄脏外胚层，体节，脊索，以便于识别和定位。 </li><li>着眼于前部的胚胎脊索，小心地取出任何剩余的中胚层，暴露的大脑和视神经囊泡（ 图2C）。 </li><li>一旦大脑和视神经囊泡完全暴露出来，用钳子切断后方的大脑的神经管及相关外胚层。 </li><li>打开这部分（大脑和视神经囊泡）使外胚层之上，。 </li><li>小心不要损坏底层的光纤泡，仔细地重新移动的外胚层（ 图2D）。 </li><li>最后，使用镊子，视神经囊泡分开从大脑（ 图2D和2E）。 </li></ol> 台25岁以上的胚胎： <ol start="23"><li>虽然胚胎是在麻醉板 ，用钳子取出的胚胎的腹侧部分。 </li><li>为了更好地对比，将胚胎到60毫米的塑料培养皿中含有10毫升的0.1倍MMR和100微升1％的硫酸耐尔蓝2-3分钟，然后转移到夹层板 。这会污染外胚层蓝色。 </li><li>一旦在夹层板 ，拉回来的外胚层覆盖的视网膜（或视泡在胚胎阶段26-30）（ 图2F和2G）。耐尔蓝硫酸盐，外胚层将被染成蓝色，但相关的视网膜组织不会。 </li><li>小心取出视网膜或视泡，以forceps（ 图2H，2I，2J）。是有帮助的按住胚胎到位用1双镊子，和删除与其它视网膜或视神经囊泡。 </li></ol> 2。解离组织和细胞的电镀 重要提示 -当转移组织，不使视网膜或视神经泡触及任何气-液边界，如果发生这种情况的细胞裂解。 <ol><li>视泡或视网膜解剖后，小心转移到外植体 ，， 冲洗板与P100微量移液器使用气雾剂抗提示，并允许坐，持续30秒。 </li><li>将80微升0.1％胰蛋白酶CMF从外植体的解离板的外植体解离地铁 。 </li><li> P100微量移液器和气溶胶性提示使用，转让视网膜或视泡从外植体冲洗板外植体Dissociati转让的外植体管，避免冲洗的解决方案。 </li><li>允许的组织的外植体的解离管中离解，在室温下的1小时。一个视网膜每块板使用，但是，我们注意到，如果需要的话，以增加在每块板的细胞数量，可以使用一个以上的每板。 </li><li>板的细胞，先慢慢取出的外植体解离地铁 40微升的解决方案，并放弃使用P100微量移液器和气溶胶性提示。使用P100集40μl和气溶胶耐提示，将细胞组织分离培养板 。当吸取细胞到板，移液缓慢，并保持在板的中心，在一个小区域的细胞。 </li></ol>注意：如果整个实验过程中是将要采取的多个细胞的图像，附加一格（Cellattice）的培养板的底面</STRONG&gt;一小滴胶水。这将允许在相同的方向中相同的细胞图像时须采取的过程中的不同点。 <ol start="6"><li>允许细胞以静置至少1小时，坚持Nunclon板。在此之后，他们必须非常轻柔的对待，他们可能会成为分离。 </li><li>培养细胞，通过观察同级胚胎，饲养细胞在相同温度下作为所需阶段。如果活细胞不被用于进一步的操作，就可以在这个时候固定在MEMFA溶液。 </li></ol>重要注意事项：我们的实验是固定的6小时内电镀的细胞。在培养的细胞是长寿取决于在哪个阶段的组织解剖和蛋黄仍然存在于细胞的量;从年轻（神经胚阶段）细胞解剖保持5-6天，而细胞获得健康的文化老胚胎（游泳的蝌蚪阶段）保持有效的培养2-3天。 3。钙成像47-49 该协议利用钙敏感Fluo4-AM和共聚焦显微镜定量分析单个细胞的钙瞬变。应执行的所有步骤使用Fluo4-AM避光，作为Fluo4-AM是光敏感。所有洗涤应添加或删除一个P1000微量移液器及防提示。移液应做得缓慢和朝向边缘的板，以避免干扰细胞的。媒体的文化没有改变，因为一般是固定的6小时内被解剖。长期的文化，媒体应每天更换。 <ol><li> fluo4-AM被储存在-20℃下，我们建议存储于5μl等分试样。在使用之前，解冻5微升等分Fluo4-AM避光，并加入2μl的Pluronic F-127直接到这等份。 </li><li>培养后的细胞为所需量的时间（至少了1小时）中，取出1毫升的细胞从培养板的培养基中。这Fluo4-AM和Pluronic，混合温柔移液。 </li><li>慢慢所有该溶液转移到要成像的细胞培养板的边缘，并留下的细胞，在该溶液中，静置1小时至吸收的Fluo4-AM。 </li><li>要洗出多余的Fluo4-AM细胞培养液中，完成下列洗系列： <ul><li>删除板1毫升的媒体。 </li><li>加入3毫升新鲜的细胞培养介质中（从15毫升锥形管）的板。 </li><li>执行两个额外的洗涤液，每次慢慢从板中除去3毫升溶液，并加入3毫升新鲜的细胞培养介质。 </li></ul></li><li>将细胞现在应该在4毫升的细胞培养基和包含Fluo4-AM。 fluo4-AM酶解一旦进入细胞，以防止它从扩散出来的细胞或成任何膜结合舱室。 </lI&gt; <li>共焦显微镜的成像搬上舞台，在加载板，画一条垂直的红色线盘上的永久性标记，同时盖，以防止标记颗粒污染的文化。的垂直线向下绘制的陪替氏培养皿（的基础上的陪替氏培养皿，而不是盖）的一侧。其目的是为了指示的方向相对于视场的培养板的各个细胞的精确定位和对准，以便可以重新建立在稍后的点。 </li><li>现在已经准备好装走上舞台的共聚焦显微镜成像板。可视化的细胞，用明视场设置在激光扫描共聚焦显微镜（物镜20X）。成像时，发现一处茂密的细胞不包含团块，最好有一个字段的视图，其中包含的大电网的数字上的Cellatice的盖玻片的观点更容易找到相同的领域后，在SI的文化TU杂交。在成像之前，通过培养和重心下移采取明亮视场图像的记录格下面的细胞的细胞培养物的位置和取向。这允许调整，用于随后的分析的细胞。 </li><li>提高焦平面捕捉亮场图像的细胞开始的钙成像（ 图3A）。 </li><li>一旦细胞的中心平面是在焦点，该板是准备钙活性的成像。设置会有所不同，这取决于显微镜和应用程序。我们的形象的使用氩488 nm激光的细胞为1，2，或12小时的使用下面的参数： <ul><li> 1小时的图像 ： <ul><li>氩气488 nm激光扫描，其最大的30 mW的功率在4％左右。 </li><li>扫描一次，每4秒（900扫描每小时）。 </li></ul></li><li> 2小时图像： <ul><li>氩气488 nm激光扫描，其最大的30 mW的功率在4％左右。 </li><li>扫描一次，8秒（450扫描每小时）。 </li></ul></li><li> 12小时的图像： <ul><li>氩气488 nm激光扫描，其最大的30 mW的功率为2％。 </li><li>扫描一次，每48秒（75扫描每小时）。 </li></ul></li></ul></li></ol>这些参数将导致在每个时间帧的一组900的静止帧图像。钙活性然后，可以记录使用的图像处理应用程序，如ImageJ的50以上的时间过程的图像（ 图3B）与通过分析荧光水平。 4。修复细胞<ol><li>成像，细胞可固定30分钟，通过从板中除去3毫升溶液和余下的1毫升培养基中加入1毫升的2X MEMFA到。 </li><li>固定后，取出MEMFA和脱水5分钟清洗，每一个体积为1毫升的一系列细胞： <ul><li> 1X磷酸盐缓冲盐水（PBS）中的25％的乙醇。 </li><li>在1X PBS的50％乙醇。 </li> &lt;李SDD H 2 O（无菌去离子蒸馏水）&gt; 75％的乙醇。 </li><li>在最后一次洗涤用1毫升100％乙醇中，于-20℃下的存储板更换</li></ul></li></ol>注：甲醇也可以用于这些清洗和用于存储。 5。 荧光原位杂交（FISH） 文化可以测定的爪蟾使用标准FISH协议的细胞培养的修改安德森实验室52 51。安德森实验室协议的修改在下面列出。所有洗涤液都在约1毫升体积的35毫米Nunclon板进行的，除非另有说明。解西伯等的定义53，除非另有说明。 重要注意事项：不要用荧光素标记的RNA探针进行原位杂交技术在细胞成像为活性钙。抗荧光素抗体结合残留的Fluo4-AM的细胞，并可能导致积极的信号，在所有细胞中的文化。 第1天：通透和杂交<ol><li>补液洗涤，则应分级脱水溶剂用于存储。对于我们的实验中，洗涤液分级乙醇在1X PBS。 </li><li>补液，用1X PBS洗涤细胞3次，每次5分钟，在室温下。 </li><li>混合25毫升Falcon管中的乙酸酐与62.5微升的0.1M三乙醇胺（pH8.0）中，并迅速混合直到完全分散安德森实验室52协议中讨论。在该混合物中，在室温下的10分钟的孵育培养。乙酸酐处理后，洗涤细胞1X盐水柠檬酸钠（SSC）中，在室温下5分钟。 </li><li>移除最后的SSC洗涤，用0.2M HCl（sdd的H 2 O）中替换为10分钟至透性细胞。/ P&gt; <li>洗出盐酸有两个用1X PBS洗涤5分钟。 </li><li> Prehybridize在ISH缓冲区中最低6小时在振荡水浴中，在60℃下，但不prehybridize过夜，因为这会严重削弱信号。 </li><li>为8-14小时，在750微升稀释的探针（1:250稀释从纯化的探头）在60℃下杂交过夜。将纯化的探针浓度范围从1.0-1.5微克/微升。 </li></ol>第2天：去除未结合的探针和抗体反应<ol start="8"><li>取出探头和冲洗培养5分钟在0.2X SSC在60℃下在新鲜的0.2X SSC洗在60℃下1小时</li><li>删除的培养从水浴中，并允许他们调整至室温5分钟。 </li><li>冲洗在0.2X SSC在室温下5分钟。 </li><li>在1X PBS洗15分钟，用0.​​1％的Triton-X-100（PBT）。 </li><li>要灭活内源性过氧化物酶，洗1小时，在2％的H 2 O 2的溶液中PBT。 </li><l&gt;冲洗用0.1％Tween 20（TBST）的与一个15分钟的洗涤1X的Tris缓冲盐水。 </li><li>座在马来酸缓冲器（MAB）在2％封闭试剂，在室温下的1小时。我们已经发现，这种阻塞的方法增加灵敏度在以前的方法相比，在20％羊血清在PBT的阻挡。 </li><li>在4℃下孵育培养在1毫升2％封闭试剂含有1:1,000稀释的POD抗体，过夜，在人与生物圈</li></ol>第3天：去除未结合的抗体和荧光发展<ol start="16"><li>移除抗体溶液，并在TBST冲洗3次，在室温下5分钟。 </li><li>在TBST中洗涤4次，在室温下的15分钟，同时连续地摆动以缓慢的速度。 </li><li>在PBT中洗两次，在室温下10分钟。 </li><li>应用1:200荧光素Cy3的酪胺或1:25的酪胺稀释在PBT的750微升。在室温下孵育5分钟，并添加2.5微升0.3％的H 2 </sub&gt; O 2。岩石为40分钟，使信号发展。 </li><li>至少4次洗净，每次15分钟在TBST中在室温下与摆动。 </li><li>一旦信号已全面开发，在1X PBT洗为5分钟。 </li><li>在4℃下1小时，在室温下，并存储在1X PBS修复在1X MEMFA为</li></ol> 6。图片FISH结果和有限公司注册与钙成像数据<ol><li>的FISH完成后，细胞培养板进行成像，以确定哪些细胞显示出积极的荧光信号，对于一个给定的探针。使用Cellattice盖玻片准确地找到所需的视场在钙成像研究，并对齐的板的取向的钙的图像帧。 </li><li>关注的中心面的细胞，并采取单一的高清晰度图像，使用氦氖氦氖543 nm激光的15％，其最大的1 mW的功率（ 图3C）。 </li><li>在最初的900帧拍摄的图像钙成像协议，识别单个细胞的感兴趣区域（ROI的），通过使用图像处理程序（例如如ImageJ的50）。提取的ROI的荧光数据作为一组，表示对时间的荧光值（ 图4）的数据点。 </li><li>叠加的FISH结果图像，从钙的图像（ 图3D）与所识别的感兴趣区。 </li><li>注册每个ROI为阳性的探针，探针阴性，或未知的 - 在对应的单元格的ROI的情况下，可以不再被发现FISH图像的视图内的字段。 </li><li>过程的投资回报率荧光数据，利用统计分析脚本（通过MATLAB或其他编程语言的设计），比较在不同的解剖阶段（ 图5A，5C）或不同的FISH探针（ 图5B和5D）阳性细胞的活性钙水平。在我们的实验室中使用的所有脚本是自由可应要求提供。 </li></ol>

没有利益冲突的声明。

凭借其良好的特点是保守的细胞类型，在所有脊椎动物中，视网膜提供了一个有用的模型研究细胞的分子过程规范和分化细胞类型。原代细胞培养提供了一个功能强大的方法，调查范围广泛的过程，包括基因表达，蛋白质动力学，钙和电活动在单细胞分辨率水平。在这里，我们提出了一个简单的原代细胞培养技术，从解剖假定视网膜组织在非洲爪蟾 ，特别适合生物体进行此类研究的假定，...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td></td></tr><tr><td></td><td></td><td></td><td> 对于解剖和培养 </td></tr><tr><td> BD猎鹰易握组织培养皿，35毫米</td><td>费舍尔</td><td> 08-772A </td><td></td></tr><tr><td>一次性聚苯乙烯培养皿中，60 x 15毫米</td><td>费舍尔</td><td> 0875713A </td><td></td></tr><tr><td> 35毫米Nunclon表面的培养皿（与Airvent） </td><td>费舍尔</td><td> 12-565-91 </td><td></td></tr><tr><td>蒙特精钳（Dumostar＃55） </td><td>费舍尔</td><td> NC9341917 </td><td></td></tr><tr><td> Cellattice微统治塑料盖玻片，25毫米</td><td>费舍尔</td><td> 50-313-17 </td><td></td></tr><tr><td>甲烷磺酸乙基 - 间 - 氨基苯甲酸盐（MS-222） </td><td> MP Biomedicals，LLC </td><td> 103106 </td><td></td></tr><tr><td>硫酸庆大霉素</td><td>恩佐生命科学</td><td> 380-003-G025 </td><td></td></tr><tr><td> Gibco公司的胰蛋白酶（1:250）粉末</td><td> Life Technologies公司</td><td> 27250-018 </td><td></td></tr><tr><td> 溶组织梭菌胶原酶B从</td><td>罗氏公司</td><td> 11088831001 </td><td></td></tr><tr><td>青霉素 - 链霉素</td><td> Gibco公司</td><td> 15140-122 </td><td></td></tr><tr><td>耐尔蓝硫酸盐（可选） </td><td> Pfaltz＆鲍尔</td><td> N05550 </td><td></td></tr><tr><td> 500毫升真空过滤机/存储瓶系统，0.22μm孔径33.2平方厘米 CN膜</td><td>康宁</td><td> 430758 </td><td></td></tr><tr><td></td><td> &lt;/ TD&gt; <td></td><td> 对于钙成像 </td></tr><tr><td>氟凌晨4点1毫米的DMSO溶液，细胞永久</td><td> Life Technologies公司</td><td> F-14217 </td><td></td></tr><tr><td>的Pluronic F-127 10％水溶液</td><td> Life Technologies公司</td><td> P-6866 </td><td></td></tr><tr><td> LSM 510共聚焦显微镜系统</td><td>蔡司</td><td>型号停产</td><td></td></tr><tr><td>阻断剂</td><td>罗氏公司</td><td> 11096176001 </td><td></td></tr><tr><td></td><td></td><td></td><td> 对于荧光原位杂交（FISH） </td></tr><tr><td>抗地高辛过氧化物酶（POD），Fab片段</td><td>罗氏公司</td><td> 11207733910 </td><td></td></tr><tr><td>抗-DNP-HRP抗体</td><td> Perkin-Elmer公司</td><td> NEL747A </td><td></td></tr><tr><td> CY3NHS酯</td><td> GE医疗集团</td><td> PA13101 </td><td></td></tr><tr><td> NHS-荧光素</td><td> Thermo Scientific的</td><td> 46409 </td><td></td></tr><tr><td>甲酰胺，去离子水</td><td> AMRESCO </td><td> 0606-950毫升</td><td></td></tr><tr><td> Torula RNA，IX型</td><td> Sigma-Aldrich公司</td><td> R3629 </td><td></td></tr><tr><td>肝素钠盐，从猪小肠粘膜</td><td> Sigma-Aldrich公司</td><td> H3393-250KU </td><td></td></tr><tr><td> CHAPS </td><td> Sigma-Aldrich公司</td><td> C3023 </td><td></td></tr><tr><td></td><td></td><td></td><td> 表2中。具体的试剂和设备。 </td></tr><tr><td></td><td></td><td></td><td><table border="1"><tbody><tr><td> 解 </td><td> 参考 </td><td> 内容 </td></tr><tr><td>细胞培养液</td><td>张和斯皮策2009年54 </td><td> 116毫米的NaCl 0.67毫米氯化钾 2毫米氯化钙 1.31毫米硫酸镁 4.6毫米的Tris 1％（体积比）青霉素/链霉素 用盐酸调节pH值至7.8。 通过0.22μm的CN过滤器过滤灭菌。 存放于4℃ </td></tr><tr><td>钙，镁培养基（CMF） </td><td> Gu 等人 ，1994年42谷和斯皮策，1995年55 </td><td> 116毫米的NaCl 0.67毫米氯化钾 4.6毫米的Tris 0.4 mM的EDTA 1％（体积比）青霉素/链霉素 用盐酸调节pH值至7.8。 通过0.22μm的CN过滤器过滤灭菌。 存放于4℃ </td></tr><tr><td>马来酸缓冲液（MAB） </td><td>西伯等人，2000年53 </td><td> 100毫米马来酸 150mM的NaCl（pH 7.5）中。 </td></tr><tr><td>马克的改良林格（MMR），10X </td><td>西伯等人，2000年53 </td><td> 100mM NaCl的 mM KCl中 1毫米硫酸镁 2毫米氯化钙 5毫米HEPES 用NaOH调节pH至7.4 0.1X MMR还含有50毫克/毫升的硫酸庆大霉素和pH用NaOH调至7.4。 </td></tr><tr><td> MEMFA解决方案，10X </td><td>西伯等人，2000年53 </td><td> 0.1 M MOPS（pH值7.4） 2毫米EGTA 1毫米硫酸镁 3.7％的甲醛 甲10X溶液，无甲醛，可以被存储在4℃下甲醛加入新鲜的（1/10体积的一个标准的37％的股份）。 </td></tr><tr><td> 原位杂交缓冲液</td><td> ·西夫E 等人，2000年53 </td><td> 50％甲酰胺 5X SSC 1毫克/毫升Torula核糖核酸 100毫克/毫升肝素 1X Denhart的解决方案 0.1％吐温20的 0.1％CHAPS的 10mM的EDTA。 </td></tr></tbody></table> 的解决方案。*庆大霉素，抗生素，用于在我们的MMR解决方案，而在我们的培养基使用青 ​​霉素和链霉素。 </td></tr></tbody></table>

dissection, culture, and analysis of xenopus laevis embryonic retinal tissue

前区的神经板产生的脊椎动物视网膜的过程仍然是一个重大的临床和基础研究的重点。除了 ​​明显的理解和治疗视网膜疾病的医疗相关的脊椎动物视网膜的发展作为一项重要而优雅的模型系统的理解决定和分化的神经元细胞类型1-16。的视网膜神经由6个独立单元（神经节细胞，无长突细胞，水平，感光细胞，双极细胞和Müller胶质细胞），其布置在定型层，一个模式，主要是保守的所有脊椎动物12,14-18。 在学习的完整胚胎发育的视网膜中明确要求的理解这个复杂的器官如何发展一个突出的前脑的层状结构，有很多问题，有利于从米采用采用推定视网膜细胞的原代细胞培养7,19-23。例如，分析细胞组织去除和分离在不同的阶段可以辨别的状态规格的单个细胞在不同的发展阶段，也就是细胞的命运的情况下与周围组织的相互作用8,19-22 ,24-33。原代细胞培养也允许调查员来处理与特定的试剂和文化上单个细胞水平5,8,21,24,27-30,33-39的，分析的结果。一个经典的模型系统，研究非洲爪蟾，早期神经发展19,27,29,31-32,40-42的 ，作为一个特别合适的视网膜原代细胞培养系统10,38,43-45。 推定视网膜组织从最初的发展阶段，紧随神经诱导25,38,43。此外，由于日胚胎在每个单元格中包含的供给蛋黄，视网膜细胞的培养可以在一个非常简单的定义的介质组成的一个缓冲的盐溶液中，从而消除了混杂影响的潜伏期或其他血清型产品10,24,44-45 。 然而，从周围组织和随后的处理的视网膜组织的隔离是具有挑战性的。在这里，我们提出了一种方法，在非洲爪蟾（Xenopus laevis）的视网膜细胞，将被用于制备原代细胞培养，反过来，在单一细胞的分辨率的的钙活性和基因表达进行分析的解剖和解离。虽然本文介绍的主题是自发性钙瞬变分析，该技术广泛适用于广泛的研究问题和方法（ 图1）。

成功解剖视神经的囊泡（阶段25）和视网膜（阶段35）的实施例示于图2E和2J。虽然这种协议可以用于在不同的开发阶段，这是至关重要的，从而只获得视网膜组织确保准确性的进一步的实验。小心地去除表皮的各个阶段，并确保您的镊子不要刺穿视网膜组织。 35岁或以上在第二阶段中，透镜可以看到作为一个透明层的顶部的视网膜和可以由谨慎刮削使用镊子除去。 <p class="jove_content...

Watch this Scientific Journal Video about Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue at JoVE.com

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

非洲爪蟾提供了一个理想的模型系统，为研究细胞命运和生理功能的个人视网膜细胞的原代细胞培养。在这里，我们提出了一种技术，用于解剖视网膜组织并产生成像的原代细胞培养活性钙和原位杂交分析。

解剖，文化与分析<em&gt;非洲爪蟾</em&gt;胚胎视网膜组织

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

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Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

13.8K 次观看.  College of William and Mary. 非洲爪蟾提供了一个理想的模型系统，为研究细胞命运和生理功能的个人视网膜细胞的原代细胞培养。在这里，我们提出了一种技术，用于解剖视网膜组织并产生成像的原代细胞培养活性钙和原位杂交分析。

视频: Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection和胚胎小鼠海马和皮层神经元的原代培养

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

解剖鼠标眼的视网膜色素上皮的全山

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using lectin are comparable to that of the p75NTR antibody. Motoneurons from the embryonic spinal cord can be isolated by this method, survive and grow out neurites.

An alternative way of isolating mouse embryonic motoneurons from the spinal cord is described. The method takes into account the fact that lectin can bind to the low affinity nerve growth factor receptor p75NTR. This lectin-based preplating allows a purification similar to that with a specific antibody against the p75NTR.

基于凝集素的分离和培养的小鼠胚胎运动神经元

Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

大鼠胚胎神经细胞的分离和培养：一个快速议定书“

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

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 ...

Isolation and Culture of Endothelial Cells from the Embryonic Forebrain

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two vascular networks of the embryonic forebrain, pial and periventricular, are spatially distinctive and have different origins and growth patterns. Endothelial cells from the pial and periventricular vascular networks have unique gene expression profiles and functions. Here we present a step-by-step protocol for isolation, culture, and verification of pure populations of endothelial cells from the periventricular vascular network (PVECs) of the embryonic forebrain (telencephalon). In this approach, telencephalon devoid of pial membrane obtained from embryonic day 15 mice is minced, digested with collagenase/dispase, and dispersed mechanically into a single cell suspension. PVECs are purified from cell suspension using positive selection with anti-CD-31/PECAM-1 antibody conjugated to MicroBeads using a strong magnetic separation method. Purified cells are cultured on collagen 1&#160;coated culture dishes in endothelial cell culture medium until they become confluent and further subcultured. PVECs obtained with this protocol exhibit cobblestone and spindle shaped phenotypes, as visualized by phase-contrast light microscopy and fluorescence microscopy. Purity of PVEC cultures was established with endothelial cell markers. In our hands, this method reliably and consistently yields pure populations of PVECs. This protocol will benefit studies aimed at gaining mechanistic insights into forebrain angiogenesis, understanding PVEC interactions, and cross-talks with neuronal cell types and holds tremendous potential for therapeutic angiogenesis.

This video demonstrates an easy and reliable strategy for preparation of pure cultures of endothelial cells from the embryonic forebrain within 10-12 days and will be useful for research focused on many aspects of cerebral angiogenesis.

从胚胎前脑内皮细胞的分离和培养

Embryonic brain endothelial cells can serve as an important tool in the study of angiogenesis and neurovascular development and interactions. The two ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

一个简单的行为分析在检测视觉功能<em&gt;非洲爪蟾</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

通过的嗅球钙监控录像的变化温度引起的神经元电活动<em&gt;非洲爪蟾</em

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

爪蟾Tectal 神经元全细胞膜片钳记录的制备与协议

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...