由美国国立卫生研究院授予的HD - 32116，桑德勒家庭支援基金会，约翰宝干细胞基金，文部科学省，厚生劳动省和HFSP支持的工作。 ZM的支持卡洛斯Baldoceda基金会和加州大学旧金山分校Krevans的奖学金。 AA - B的。持有希瑟和Melanie搞乱主席赋予。

一，准备与室管膜流量检测的荧光微球（可以跳过这些步骤，如果只准备染色的目的wholemounts）填充玻璃微量移液器。 <ol><li>一个Wiretrol 5 UL玻璃毛细管玻璃微管拉拔到安全和调整加热器和电磁设置拉带吸管光滑，浅锥。 </li><li>连接正气压源上拉微量，轻轻地放下一个金属光栅表面上的吸管尖成45 °角，以创建一个斜角的提示。正压差，有助于清除玻璃碎片从吸管内。清洁的乙醇蘸组织的斜面提示。 </li><li>检查微米的显微镜下一个吸管尖。提示〜1​​00微米的内部开口直径应该有一个光滑的斜面。可用于直径较小，但往往会造成堵塞吸管用荧光珠。 </li><li>回填吸管与矿物油到半满，然后将其插入吸管回油脂浸柱塞。提前半月板的矿物油通过手动推动柱塞的枪头。 </li><li>到显微安全微管和活塞，然后到一个小螺丝可调高度的固定臂显微吸管持有人。 </li><li> Frontload与荧光微珠荧光微珠原液50％，45％水，5％的甘油溶液组成，吸管。添加甘油，以增加密度的解决方案，使沉积到wholemount微球下沉到表面。 </li><li>放置在一个安全的地方，针不会被意外损坏，着手wholemount清扫的显微操纵器。 </li></ol>二。 Wholemount解剖和固定<ol><li>要准备wholemount清扫，温暖的足够数量的L - 15维茨媒体37 ° C。您将需要约10毫升，每头动物，你打算剖析。还收集所有您需要的体视显微镜解剖和固定的供应：剪刀，大齿钳，光滑细腻钳，Sharpoint 22.5 °显微捅刀，解剖盘，纸巾，生物危害袋，和24孔板上冰充满4％多聚甲醛，0.1％的Triton X - 100的带或不带。 TRITON X - 100是用来降低表面张力的煤灰的解决方案，从而降低剪切wholemount表面时，沉浸在这个解决方案的发病率。 </li><li>倒入放在一个荧光体视显微镜下解剖菜5-10毫升的回暖媒体。解剖菜准备浇筑成一个6厘米的塑料盘的弹性聚合物，称为Sylgard 184，并让聚合物溶液在真空条件下1周治愈，然后彻底冲洗大量的水在使用前菜。通常情况下，我们让菜浸泡在1升烧杯中水1周。 </li><li>动物是由颈椎脱位处死，头部被切断。 注意：如果需要的话，可能是血液从血管中清除所用生理盐水灌注动物大脑解剖前。如果执行与二氨基联苯胺（DAB）显色免疫，这一点尤为重要。 </li><li>中线切口，后到前，沿头皮揭示的头骨。 </li><li>一系列削减在颅骨打开头盖骨：一切跨越两个轨道前的嗅球，未来两年削减不如小脑和独立的头盖骨，颅底，并晋级决赛运行后到前沿矢状缝中期。 </li><li>颅瓣轻轻地缩回，大脑中提取，放在解剖盘。 </li><li>剥离剩余的体视显微镜下进行。首先，嗅球解剖远离大脑。如果你想此外，研究嗅球，只需修复它们浸泡在4％的煤灰过夜，随后你可能准备切片和染色。 </li><li>除以沿大脑纵裂。 </li><li>面向一个冠状切口在最后纵裂方面，允许尾海马可视截面。 </li><li>海马，这形式在这个位置上的侧脑室的内侧壁，然后必须覆皮质释放，形成脑室背侧壁。首先，刀插入小心室之间的皮层和海马背部空间，并削减在皮层，它反映了腹部，远离中线，加入海马。削减后，可以慢慢去皮皮质远离揭示移动从背到腹侧脑室的海马。这一演习是切断海马被释放的角落的皮质楔形加快。达到在这个位置上的侧脑室腹侧最大程度后，您可以直观感觉皮层再次环绕，此时反映背面内侧，加入海马。另一个削减必须在这个位置上，完全释放海马或从皮质或侧脑室外侧壁侧脑室的内侧壁。 </li><li>然后它会很容易拉离皮层海马，内侧和前方，打开侧脑室广泛。 </li><li>继续轻轻一拉前方用小镊子和刀招收回的内侧和外侧墙壁除了海马。 </li><li>一旦这个回缩的阻力开始增加，需要额外的削减。第一，增加您的曝光侧脑室，尤其是外侧壁和SVZ，剖析了皮层。皮质是干净的解剖，通过可视化界面之间的胼胝体和VZ / SVZ。只要沿着这条接口上停留的胼胝体一侧，以避免损坏SVZ削减。 </li><li>为了继续回缩远离侧壁内侧壁，需要两个或更多的裁员是：削减背部的侧壁，内侧壁和皮质都收敛，和一个腹部切侧壁，内侧壁和丘脑收敛。随着这些削减，进一步温柔的内侧墙壁上回缩允许侧脑室前，最大程度的开放。 </li><li>适当的照明是必不可少的整个过程中，特别是在下一步前方内侧和外侧墙壁分开。此时在侧脑室前的位置，内侧的墙壁反射回连续侧壁。调整灯光，两墙之间的投射阴影，这种反射点，像两墙之间的山谷中出现。正是在这个山谷切割分开这两个墙壁。 </li><li>最后，完全暴露删除任何悬垂背部皮层和丘脑腹侧侧壁。 </li><li>如果准备wholemounts免疫，仔细转移wholemount，心室的一面，从解剖菜，到充满了4％的煤灰过夜固定带或不带0.1％TRITON - X100 24孔板在4 ° C。对于固定敏感的抗原，wholemounts可能是固定的时间较短。然后进行第4免疫wholemounts。 </li><li>如果准备室管膜流量分析whol​​emounts，转让wholemount充满新鲜，37℃Leibovitz的中型到一个干净的清扫菜的，并继续下一节。 </li></ol>三。室管膜流量分析用荧光微球<ol><li>固定在一个干净的解剖盘使用2昆虫针，在丘脑和前背wholemount的角落之一wholemount。 </li><li>将固定旁边的体视显微镜的显微的手臂基地。确保可调手臂的高度，最大限度地提升，以避免破坏对解剖盘针。 </li><li>小心沾在ependorf管针在媒体提示清洁针的外部提示的微球。如果这些都没有洗清，他们可能随后wholemout表面上沉积，无意中在针定位，并减少影片的整体质量。 </li><li>针尖在侧壁背水面的位置和较低的手臂，使针尖进入中期。针应该降低，直到它的正上方侧壁表面。 </li><li>针位置，调整缩放的体视显微镜上，重点涵盖了所需的字段。如果您将一个录音室管膜流动，开始在这个时候获得的图像。 </li><li> 〜微珠解决方案5 NL wholemount表面上弹出。一旦初步珠丸被清除由室管膜的表面流动，可以执行额外轮珠弹射。 </li></ol>四。免疫Wholemounts <ol><li>为免疫解剖Wholemounts是浸泡固定在4％的煤灰隔夜或不0.1％TRITON - X100在4 ° C TRITON - X100的使用是首选的容忍这种治疗的抗原，但可以留在染色质量，减少洗涤剂的情况下进行。 </li><li>第二天早晨，PFA是吸气从24孔板wholemounts都在0.1 M PBS洗涤3次，每次5分钟，或0.1％TRITON - X100没有。如前所述，TRITON - X100使用的是首选，但不是必需的，在本议定书的所有洗。纵观本议定书，在整个交换解决方案安装需要小心从一侧以及解决方案的愿望。然后，以及使用过的很好，没有直接到wholemount方的角度等，清洗解决方案的移液管的溶液补充。要注意保持wholemount在任何时候都面临心室。大力吹打的解决方案往往会翻转wholemount。我们宁愿超过1 wholemount交换一次，以防止组织干燥的解决方案。 </li><li>洗涤后的煤灰，wholemounts孵育1小时，在室温下封闭液，10％正常山羊或在0.1 M PBS驴血清载有或没有TRITON - X100。如果使用您的染色TRITON - X100，您可以选择使用2％或0.5％TRITON - X100在封闭液。我们用2％TRITON - X100抗原，需要深入到组织的抗体渗透，如位于SVZ这些抗原，染色时。然而，位于接近侧壁，如在心尖部室管膜细胞表面发现的抗原，表面抗原染色时，我们使用0.5％的Triton - X100。除此之外，TRITON - X100可对细胞表面或其他被删除或者改变洗涤剂的抗原。 </li><li>接下来，去除阻塞的解决方案，并添加在同一封闭液稀释的抗体，并为24或48小时孵育4 ° C。潜伏期的选择取决于抗原，类似的选择0.5％或2％的Triton。对于位于wholemount表面抗原，24小时的潜伏期就足够了。但是，更深层次，如设在SVZ的抗原，48小时的潜伏期，提供了较好的效果。 例如，研究根尖表面与基体的衬侧脑室壁细胞，与β- catenin的抗体染色，标签细胞膜，和γ-微管蛋白，标签基体。 0.1 M的PBS含10％正常山羊血清和0.5％TRITON - X100稀释的鼠抗β- catenin的抗体（1:500）和兔抗γ-微管蛋白抗体（1:1000） 。在4 ° C孵育24小时。 染色成体神经干细胞，或键入B1细胞，GFAP抗体染色的侧壁。 0.1 M的PBS含10％正常山羊血清和2％的Triton - X100稀释的鼠抗GFAP抗体（1:500） 。孵育在4 ° C 48小时。 </li><li>主要的抗体洗掉2快速冲洗最初的PBS或无0.1％TRITON - X100。然后在室温下每20分钟做3额外的清洗。 </li><li>稀释二次抗体阻断初级抗体在同一解决方案，并添加到wholemounts初级抗体孵育相同的时间长度在4 ° C 例如，β- catenin和γ-微管蛋白染色：淡化的Alexa Fluor 488山羊抗鼠抗体（1:400，识别鼠标抗β- catenin的）的Alexa Fluor 594山羊抗兔抗体（1:400，认识到，在0.1 M的PBS含10％正常山羊血清和0.5％TRITON - X100，兔抗γ-微管蛋白） 。 在4 ° C孵育24小时。 GFAP的免疫：稀释的Alexa Fluor 488山羊抗鼠抗体（1:400，承认小鼠抗GFAP）在0.1 M的PBS含10％正常山羊血清和2％的Triton - X100。孵育在4 ° C 48小时。 </li><li>二级抗体洗掉wholemount使用初级抗体执行相同的洗涤。 </li><li>如果需要的话，核反击染色可以在这一点上，在PBS稀释的DAPI为30分钟，在室温下，然后在PBS洗涤一次孵化。 </li></ol>五，安装到幻灯片共聚焦显微镜免疫染色Wholemounts <ol><li>对于高分辨率的共聚焦成像，免疫需要分解剖，以保持其作为一个组织的200-300微米厚的银侧脑室外侧壁wholemounts。从底层纹状体侧壁分离，允许它被安装到幻灯片，并用盖玻片覆盖在一个单位的方式。 </li><li>返回的体视显微镜，免疫染色wholemounts和下面的工具和设备：光滑的细钳，Sharpoint 22.5 °显微捅刀，解剖盘，显微镜载玻片和盖玻片，0.1 M的PBS，Aquamount安装介质。 </li><li>从24孔板解剖菜含有0.1 M的PBS小心保持心室侧的wholemount传输。 </li><li>首先，从后到前完全去除切割正是沿胼胝体符合侧壁行wholemount背皮层。这是公认的接口之间的胼胝体白质和粉红色的出现SVZ。 </li><li>然后跨腹侧方面的wholemount水平方向切。这个切割面将提供一个平台上，你可以ST下一步在解剖过程中abilize wholemount。 </li><li>背水面的wholemount朝上，你将能够可视化SVZ的厚度由前，后沿侧壁。请注意，这种观点是由可能让底层的纹状体和SVZ皮层可见的初步去除。 SVZ被确定为组织的薄带从心室表面延伸到纹状体。 SVZ有均匀的粉红色外观，而纹状体渗透脑白质线。请注意，SVZ是较厚的前方，并逐渐变得更薄向后。 </li><li>一旦你已经确定SVZ和纹状体的接口，仔细开始，在此接口在最前方面的侧壁的切割，推进刀从背腹。要做到这一点准确，稳定的两个引脚用于您的镊子wh​​olemount。您也可以使用您的镊子稍微转动wholemount，以可视化的推进整个侧壁从背腹刀片。剥离的关键是对组织造成的条子非常平坦。这意味着，你片SVZ，由前至后整个侧壁削减的方向，在任何时候都心室表面必须保持平行。 </li><li>请记住，更多的后方，SVZ变得更薄。而不是变薄了剥离，切断了这一点只有在SVZ，重要的是，当你向后推进，被解剖组织的厚度保持不变。这将确保组织的条子，你也将随之装入是平坦的。 </li><li>完全分离后从底层纹状体的侧壁，小心地取出所有其他周围组织，从这个条子，不室壁的一部分。 </li><li>从下面的使用您的镊子然后拿起这个条子，并在载玻片中心的位置。请直接到wholemount aquamount几滴，轻轻放置在这个中心的盖玻片，尽量不引进到组织表面的气泡。在幻灯片的重量将确保aquamount分散均匀，会产生一个精致的扁平化的侧壁表面。使用的aquamount量和盖玻片的大小取决于被解剖组织的年龄。对于胚胎和产后早期组织，我们宁愿aquamount和1滴22“X 30”盖玻片。较重的盖玻片可能扭曲的组织，更aquamount会干扰成像质量，因为共聚焦激光将能够穿透aquamount居住之间的盖玻片和组织表面薄薄的一层。对于后产后和成人组织中，我们使用4滴aquamount和24“X 60”盖玻片。 </li><li>幻灯片，然后平放于4 ° C的成像前1-2天，以便解决的盖玻片在幻灯片书。 </li></ol>代表性的成果 Wholemount方法提供了进入成年SVZ生发活动的几个关键的见解。年轻的神经元在SVZ迁移的连锁网络首次发现后，侧脑室外侧壁wholemounts polysialylated神经细胞粘附分子（PSA - NCAM）1抗体免疫染色。这些迁移的神经母细胞链，也可以看到后免疫wholemounts doublecortin抗体（ 图1） 。值得注意的是，连锁网络有一个千篇一律的模式，与一般流的细胞，运行背部以上和腹部周围的粘连点运行。的SVZ Wholemounts还提供了一个全面的看法，在这一地区的祖细胞增殖活性与Ki67 在图 2染色。有趣的是，最近的两项研究表明除以SVZ细胞和当地的血管 2,3（ 图2）之间的密切互动。 当高功率激光共聚焦显微镜下观察，EN面对面wholemounts提供允许衬脑室系统的根尖细胞表面的一个独特的视角。这个连接面的角度来看最近透露，SVZ B1型细胞，成体神经干细胞，是与非分裂区别的4室管膜细胞混合神经上皮的一部分。 B1型细胞接触侧脑室的顶面和大心尖部室管膜细胞表面包围在风车配置（ 图3，箭头表示B1根尖表面） 。此外，心尖部室管膜细胞表面的仔细检查发现，平移的位置和旋转方向的基体是其平面极性 5指标。室管膜细胞基底博死亡都集中在根尖表面上的补丁。这个补丁是在下游“的方向发展中心从根尖表面流离失所脑脊液流（翻译极性）;在此修补程序，每个基体是其长轴旋转，这样脚的基底，基底的附件的身体，在流动方向（旋转极性）的点。邻近室管膜细胞基体在同一方向的导向。重要的是，室管膜流量检测videomicrographs可用于直接在一个特定区域的侧壁比较流室管膜细胞基底，在该地区的机构（ 图4）的方向。 除了在成人大脑的生发地区最大的全景角度，更高的功率成像，，wholemounts允许个人在SVZ细胞形态更加完整和详细的分析。高功率wholemounts免疫GFAP的共焦成像显示，B1型细胞，他们除了短期心室联系根尖过程中，有一个长期基底过程，在接触血管（ 图5）4 。这种细胞结构没有受到赞赏冠状切片以前，因为基础的进程中运行的大多是平行于室壁。因此连续切片切成小片段的单个细胞，几乎不可能重建一个细胞完整的形态，或了解它关系到其他类型的细胞在SVZ。 wholemount方法比经典切片技术的诸多优势，无论是在提供低功耗显微镜和高倍显微镜的单个细胞的完整的角度全景。这种技术将继续是一个重要的补充，这个成人脑生发区未来的研究。 <img src="/files/ftp_upload/1938/1938fig3.jpg" alt="图1" /> 图1。候鸟在SVZ神经链的网络。平铺共聚焦图像重建一个侧壁wholemount doublecortin的标签迁移整个SVZ神经母细胞抗体染色。有两个一般的移民流，运行背部以上和腹部周围的粘连点运行，由星号（*）表示。箭头指示前（a）和背侧（D）方向。比例尺= 1毫米。 <img src="/files/ftp_upload/1938/1938fig4.jpg" alt="图2" /> 图2的SVZ血管和细胞分裂之间的关系。这侧壁wholemount Ki67的抗体是免疫染色，标签对小鼠免疫球蛋白绿色，分裂细胞和抗体，标签为红色的血管。由于这wholemount是不染色前用生理盐水灌注，内源性鼠IgG分子留在血管和辅助抗鼠抗体染色。最近的工作表明，除以SVZ的前体（绿色）在位于靠近血管（红色）{沉，2008＃6523} {Tavazoie，2008＃6522}。箭头指示前（a）和背侧（D）方向。比例尺= 1毫米。 <img src="/files/ftp_upload/1938/1938fig1.jpg" alt="图3" /> 图3。心尖侧壁脑室接触细胞表面。高功率共聚焦图像一个wholemountβ- catenin的免疫染色，绿色标签细胞膜，和γ-微管蛋白，红色标签的基体，揭示了这些上皮细胞的平面组织。 B1型细胞，成体神经干细胞，有一个用一个单一的基体的小根尖表面，用箭头表示。这些细胞的顶面四周是大风车配置在心尖部室管膜细胞表面。室管膜细胞有多个根尖表面的基体的位置表示的平面极性。邻近室管膜细胞基体集群位于根尖表面的同一侧（向左向下，并在本地区），相应的脑脊液流{Mirzadeh，2010＃6573}方向。比例尺= 10微米。 <img src="/files/ftp_upload/1938/1938fig5.jpg" alt="图4" /> 图4，室管膜流量检测。合并室管膜流量检测期间采取了电影的100个连续帧创建复合图像。荧光微球敷背和后室管膜纤毛粘连面积推动了两个流，一个下粘连，朝门罗孔。这个面向流揭示室管膜细胞功能的平面极性。每个流线描绘了一个单一珠的连续时间点的位置。比例尺= 0.5毫米。 图5。GFAP + B1型细胞与基底纤维血管英尺长。高功率激光共聚焦从侧壁wholemount采取栈的最大投影标签SVZ星形胶质细胞与GFAP抗体免疫染色。染色标签成人神经干细胞，或键入B1单元格，其中有一个心室表面的根尖结束，如下所示，长GFAP +基底纤维血管（箭头）结束。因为二级抗体用于可视化鼠抗GFAP抗体识别的血管内源性鼠IgG血管染色。比例尺= 50微米。

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心室和脑室下区神经发生的大多数研究都依赖于经典切片技术研究的显微解剖，并在这些地区的蜂窝关系。在这里，我们描述一种替代技术，首先用于分析网络的迁徙产生的神经母细胞在SVZ 1链，然后用于治疗抗有丝分裂，最近用于研究的精确根尖研究SVZ祖人口再生成年SVZ神经干细胞2,3,4和基底细胞与细胞的相互作用。有趣的是，这项技术已经发现，神经干细胞，或键入B1细胞的成年SVZ，是一个有区别的室管膜细胞未分化的混合神经上皮的一部分。恩面成像wholemounts表明，这种混合神经上皮风车根尖末梢B1型包围大根尖4室管膜细胞表面的细胞组成的架构。这EN -面的分析，澄清与根尖结局，在心室的表面和接触血管利基的基础过程细胞组成的胚胎和成年大脑中的神经干细胞谱系的认识。这些研究结果已几乎是不可能的，采用经典切片技术。 Wholemounts也促进神经干细胞，通过他们接触心室心尖过程的鉴定。由于这些干细胞特异性标志物的发现，wholemounts将神经干细胞行为的识别和分析的组成部分。 侧脑室壁Wholemounts还提供了理想的角度来看，学习室管膜细胞的平面极性。室管膜细胞衬砌心室功能，以推动以协调的方式脑脊液multiciliated细胞。随着wholemount技术的室管膜上皮细胞，整个暴露的面和可染色和全面研究，从它的前，后和背腹边界。此外，室管膜流量检测进行敏锐地解剖，现场wholemounts有力展示平面偏振光室管膜纤毛产生的流量。最近的工作，使用wholemount方法已发现了5本室管膜平面极性的细胞因素。有趣的是，wholemount研究也建议，室管膜生成的脑脊液流动的chemorepellents建立梯度，引导年轻的神经元的迁移在SVZ 7。最初确定的迁徙神经链网络Wholemount的方法，因此继续提供机制，规范移民链的见解。 wholemount成像分析的VZ和SVZ增加了新的方法为双方今后的研究和方式，以澄清我们对现有研究的理解。例如，最近的一项研究表明，在成年SVZ神经干细胞与脑室8接触的CD133 + / CD24 -细胞。他们在部分免疫的基础上，这些作者声称，这些细胞multiciliated室管膜细胞亚群。然而，在我们使用的wholemount方法，它提供了更全面的整个管膜上皮的研究，我们发现，所有的室管膜细胞表达CD24和唯一心室的接触细胞，人的CD133 + / CD24 -是该类型B1的一个子集细胞4。此外，wholemount技术承诺要在未来的研究，研究神经干细胞最近描述的镶嵌在成人大脑的组织9非常有用。一些研究表明，在成人大脑中的神经干细胞是不是一个同质化的人口，但区域指定的，通常情况下可以产生嗅球interneurons只有特定的亚型。这些研究提出，可以区分神经干细胞不同亚群的特异性转录因子 10,11,12,13,14和/的表达，或由他们沿背腹和前后延伸区的区域定位侧壁9,15,16。由于区域指定的成人神经干细胞亚群的分子标记鉴定，wholemount成像应该提供一个全面的看法，这些不同的祖域沿室壁parcelation。 wholemount清扫和这里介绍的成像技术也可用于分析心室壁在胚胎。胚胎侧壁夹层，一步一步，以同样的方式执行。有只有细微的差别的难度;胚胎心室相对较大清扫容易，但组织较软，使操控更加困难。特别是，侧脑室类似的接触，可用于胚胎OS解剖心室研究皮层神经元的皮质墙。最近的证据表明，非对称中心体遗传保持桡神经胶质细胞在皮层神经元的17的心室表面。恩面的放射状胶质根尖表面成像可提供到这些分裂的细胞内中心体是如何继承不对称的见解。 与大多数技术，特别是那些涉及精确灵巧，掌握需要实践。有，然而，在解剖一个更好的结果是关键的几个要素：1）照明，调整样品的照明创建阴影提供宝贵对比在组织剥离，否则是相对均匀，2）使用像镊子从来没有使用过两个昆虫针在这项技术的钳捏在一起或拿起组织，但切割时，3）平衡的温柔回缩和切割刀不应使用机动引脚可以不断调整，以稳定组织仅可用于削减，但也提供温柔回缩至单独的内侧和外侧的墙壁，记忆，剥离的大部分实际上是通过温和的回缩只断续切削。

the subventricular zone en-face: wholemount staining and ependymal flow

侧脑室壁中含有生发在成年哺乳动物大脑中最大的地区。在这些墙的脑室下区（SVZ）是一个广泛研究的模型系统，为了解神经干细胞的行为和成年神经发生的调控。传统上，这些研究都依赖于经典切片技术进行组织学分析。在这里，我们提出了一种替代方法，wholemount技术，它提供了一个全面的，面对这生发地区。节相比，wholemounts内SVZ保存完整的细胞结构和细胞的关系。这种方法最近发现的成体神经干细胞，或键入B1细胞，是一个有区别的衬侧脑室室管膜细胞混合神经上皮的一部分。此外，这种方法已被用于研究平面室管膜细胞和脑脊液流，他们在心室产生两极分化。随着最近的证据表明，成体神经干细胞是一种异构的人口地区指定，wholemount方法，将有可能了解这种干细胞小生境的组织和parcellation的一个必不可少的工具。

Watch this Scientific Journal Video about The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow at JoVE.com

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

侧脑室壁中含有生发在成年哺乳动物大脑中最大的地区。传统上，对神经发生在这一地区的研究都依赖于经典切片技术进行组织学分析。在这里，我们提出了一种替代方法，wholemount技术，它提供了一个全面的，面对这生发地区。

脑室下区恩的脸：Wholemount染色和室管膜流量

The walls of the lateral ventricles contain the largest germinal region in the adult mammalian brain. The subventricular zone (SVZ) in these walls is an ...

the-subventricular-zone-en-face-wholemount-staining-and-ependymal-flow

Nanyang Technological University

Research

JoVE Journal

Neuroscience

41.7K 次观看.  University of California, San Francisco - UCSF. 侧脑室壁中含有生发在成年哺乳动物大脑中最大的地区。传统上，对神经发生在这一地区的研究都依赖于经典切片技术进行组织学分析。在这里，我们提出了一种替代方法，wholemount技术，它提供了一个全面的，面对这生发地区。

视频: The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

神经母细胞含量:从区分神经干细胞后代使用流式细胞仪，神经祖细胞的生成和富集含量

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

科研

神经科学

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

为揭示复杂的大脑地形wholemount免疫组化

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

钙成像的急性下区片的制备

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

新生儿脑室下区电穿孔

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile stimulation is a positive early experience that mimics maternal licking and grooming in the rat. Exposing rat pups to this positive experience can be completed easily and cost-effectively by using highly accessible materials such as a household duster. Using a cross-litter design, pups are either stroked or left undisturbed, for 15 min, three times per day throughout the perinatal period. To measure the neuroplastic changes related to this positive early experience, Golgi-Cox staining of brain tissue is utilized. Owing to the fact that Golgi-Cox impregnation stains a discrete number of neurons rather than all of the cells, staining of the rodent brain with Golgi-Cox solution permits the visualization of entire neuronal elements, including the cell body, dendrites, axons, and dendritic spines. The staining procedure is carried out over several days and requires that the researcher pay close attention to detail. However, once staining is completed, the entire brain has been impregnated and can be preserved indefinitely for ongoing analysis. Therefore, Golgi-Cox staining is a valuable resource for studying experience-dependent plasticity.

This paper describes the procedures for tactile stimulation of rat pups and subsequent Golgi-Cox staining of neuronal morphology. Tactile stimulation is a positive experience that is administered in the perinatal period by stroking pups with a household duster. Golgi-cox staining is a reliable procedure permitting the visualization of entire neurons.

可视化的一个积极的早期经验，触觉刺激的影响，对树突状形态和突触连接与高尔基染色考克斯

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

在Wholemount大鼠视网膜免疫组化和钙成像方法

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

完全性脊髓损伤和脑解剖协议后续Wholemount<em&gt;原位</em&gt;杂交苗种海七鳃鳗

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

室管膜纤毛在小鼠脑侧脑室实时成像

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

神经干细胞和祖细胞来自成年小鼠脑室下区细胞分选和实时成像的细胞周期动力学

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

分离和成人神经干细胞的培养从鼠标Subcallosal区

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...