这项工作是由校内的研究在健康/ NHLBI（1ZIAHL006126）和Lenfant生物医学博士后奖学金的颁发给DAL民族院校划分的支持。

1，所需材料的制备植三龄幼虫脑<ol><li>准备所需的显微切割工具。 <ol><li>锐意2解剖工具（手术刀和钩），首先将单个解剖针向每个引脚架。用手或钳（ 图1C）固定销紧密。 </li><li>使用一对老镊子弯曲1解剖针到约120度角。为此在解剖显微镜下，以确保销的上半部分平放时，该引脚保持器在手持。该工具将被用作手术刀按住或切片穿过组织。 </li><li>使用一对老钳子弯曲第二销到将被用于保持并刮掉组织钩。 </li><li>顶盖采用了枪头保护工具不受损坏每个解剖工具。小心，可无限期用于解剖结构良好的工具。更换损坏或变形的引脚是必要的。 </li></ol> &lt;/ LI&gt; <li>准备解剖媒介。 <ol><li>在组织培养罩，淡化抗生素 - 抗真菌剂补充到施耐德的培养介质中。漩涡媒体纳入补充。 </li><li>准备补充媒体的一些每份5毫升。存储所有媒体在4°C，直到准备使用。 </li><li>暖5ml的等分部分补充的培养基中至室温。绘制介质进入无菌5毫升注射器，拧上一个无菌的0.2微米注射器过滤器。 </li></ol></li><li>选择幼虫解剖。用解剖探头选择爬三龄幼虫的大脑将最简单的剖析。可选:存款幼虫在葡萄琼脂平板。 注:请不要使用过于拥挤瓶或瓶子与"湿的食物，"因为这些往往迫使第二或第一龄幼虫爬过早出来。 注意:某些遗传背景，将需要使用更年轻的幼虫。这不影响本协议，但不使对dissection更加困难。 </li></ol>中枢神经系统的2。解剖<ol><li>在一个单一的载玻片，由创建3厘米左右分开温暖解剖媒体的50mL池。一池将用于建筑夹层和其他用于精细解剖（ 图1C）。 </li><li>几个幼虫转印到介质滴之一。 </li><li>移动与幼虫的盖玻片解剖显微镜。放大到含有幼虫，使幼虫的前部和后部端部是可辨的池。 </li><li>使用对镊子抓住单个幼虫的中间区域。花一秒钟钳子在另一只手抓住幼虫接近相同的地区。将两个双钳拆开来轻轻地撕了一半的幼虫。重复上述两个步骤盖玻片上所有的幼虫。 </li><li>由他们转移到一个组织处置幼虫体的后半部。 </li><li>放大成幼虫，使日ê前mouthhooks都清晰可见。同时使用双钳创建一个小的切口，或缺口，在第三胸椎和第一前锯齿带之间的幼虫mouthhooks的两侧。 </li><li>把握mouthhooks与在非优势手钳。用另一只手的镊子轻轻剥离角质层中的前到后方向上。继续缓慢剥离角质层，直到中枢神经系统暴露。 </li><li>通过撕裂组织的钳子从幼虫的其余部分隔离的中枢神经系统。请小心不要扰乱中枢神经系统。 关键的一步:不要拉扯中枢神经系统！丢弃损坏的样品撕裂腹神经索（ 图2A）或扭曲视叶（ 图2B和2B&#39;）。 </li><li>重复上述两个步骤盖玻片上所有的幼虫。传送健康移植组织的介质对盖玻片上为细末解剖第二阶段（清洁）池。 </li><li>用解剖针工具清除远离大脑的外周组织（ 如眼睛，翅膀，腿和光盘）。滑动希望（脑）和不需要的组织之间的手术刀，寄托结缔组织的玻璃罩。使用钩轻轻挑逗掉不需要的组织，而同时按下手术刀盖玻片在锯般的动作。 </li><li>关键的一步:工作慢工出细活，从每个大脑取出所有碟片。请小心不要扰乱大脑的形态。一个健康的大脑都会有一个完整的腹神经索和对称，圆形视叶（ 图2C）。 </li></ol> 3，标准安装取出的大脑进行实时成像<ol><li>倒置50 mm的透气性培养皿朝上（ 图2D）的透明膜。压下注射器沉积介质的一小滴（约30微升），涂于T他心的菜。 </li><li>分离的大脑，一次一个，转移到介质上的菜的下降。使用挂钩工具舀起一个脑视叶的下面。或者，使用镊子抓住轴突从腹神经索伸出。 </li><li>收集5-10大脑在介质的压降。通过使用解剖针工具个人的大脑推到媒体的下跌，因为其中许多的底部淹没的大脑将在半月板（ 图2E）被捕获。 </li><li>用解剖针工具根据国家统计局进行成像（ 图3A），以使大脑定向的大脑。图像背侧国家统计局，地方朝下腹神经索（沿膜）。对图像中的anterio腹国家统计局，放置大脑与朝上的腹神经索（离膜）。对齐的大脑，使得所有的腹神经索在xy平面内的指向相同的方向。 </li><li>使用注射器或塑料转第ipette放置4卤烃（HC）的油滴（各约30微升）到气体可渗透膜。油每滴的体积应相当于彼此和介质的压降。空间的油的液滴，以对应于四角周围的培养介质的压降为中心的盖玻片。一旦完成，五滴（四滴的油和一滴介质在中心）将类似于五个方面对西式骰子（ 图3B）。 </li><li>降低一个＃1.522毫米盖玻片上的组件，使得它击中全部5滴在同一时间。保持均匀的压力，在整个样本减少的可能性，他们将被打乱。等待约3分钟为石油和中盖玻片的重压下散去。媒体交叉状将形成（ 图3C和3D）。 </li><li>关键的一步:通过去除用纸巾灯芯（FIGU多余的介质放下盖玻片到大脑的表面重新3C）。这样做一边看样下的解剖范围。作为介质被移除时，盖玻片将降低。一旦停止盖玻片接触到大脑。不要过度灯芯，因为这会造成组织变形或大脑爆炸。 </li><li>如果成像背NBS（ 图3E），移动到步骤3.9。如果成像anterio腹国家统计局，盖玻片必须稍微移动（通过轻轻地用镊子轻推它）中的腹神经索小费的方向。这导致大脑旋转，这带来的脑叶与盖玻片的直接接触，以促进成像（ 图3F）。 </li><li>由农村包围盖玻片与少量卤烃油（ 图3D）的密封腔。多余的油从盖玻片渗出应尽量减少，并用纸巾吸干了。 </li></ol>中脑的神经母细胞4。实时成像注:对于这项工作，尼康的Eclipse钛旋转盘共聚焦显微镜（倒置显微镜）用于实时成像;我们设置的细节此前已公布的31。可替换地，样本可以使用一个直立系统进行成像。 <ol><li>加一滴浸油的盖玻片刚刚安装的大脑以上。这将有助于中心的目标样本的正上方。 </li><li>关键的一步:在25℃的恒定温度的舞台孵化器保持大脑。安装的样品轻轻地放置到舞台孵化器，盖上盖子其室。 </li><li>找到大脑中枢的NB用透射光通过专注于驻留在视叶中部和内侧区域的大，圆形细胞， 不要使用落射荧光 。这将成为与实践更加容易。这是很重要的，该样品暴露于最少量的光成为可能。不要浪费被损坏一次成像的大脑。 </li><li>一旦到位，采取快速曝光利用共聚焦到确定适当的位置和深度。限制深度的第一10-15微米。根据需要进行调整，并采取另一种测试图像。 <ol><li>可选步骤:收集腹神经索的测试影片，以确保没有XY漂移。如果漂移被检测到，等待15-30分钟的样品来解决。如果漂移不能在30分钟解决，准备一个新的样本。主要漂移通常可以追溯到加入过量的油，或油的大小不等的安装过程中下降。 </li></ol></li><li>为了消除轴向漂移（Z-漂移）使用了一个红外LED的连续自动对焦装置。另外，手动校正焦平面。 </li><li>一旦感兴趣的NB是确定的，进行成像方案。如同所有的活细胞成像，激光的光量，曝光时间，相机的分级，物镜放大倍数，时间和/或z步间隔都必须为特定的实验手头回答的问题来优化经验。我们的目标是尽量减少光水坝年龄，同时还收集有用的数据。有关其它指导讨论。 <ol><li>图像中的NB的通过收集由1微米的z轴隔开12-14图像的整个体积。调整时间分辨率来捕获同一NB的单个或多个细胞周期。作为一般指引，用10-30秒的时间间隔为单细胞周期成像和1-3分钟的间隔为多种细胞周期。 </li></ol></li><li>关键步骤:确认样品是健康的监测有丝分裂的持续时间。如果有丝分裂时间超过15分钟，就移动到另一个大脑。一个健康的大脑会的同一视场中显示出许多的NB经历了几轮的有丝分裂。请注意，每个细胞周期略长于前一个由于光的损害。 </li><li>一次成像完成后，拆开孵化室，并放弃一切，但透气性培养的菜肴。冲洗培养皿表面用95％的乙醇中并用纸巾擦拭良好。重复2次以上。 </LI&gt; </ol> 5，制备脑的免疫<ol><li>收集20个或更多个外植大脑入1.5ml试管含有约0.2毫升补充培养基。 </li><li>从管中除去培养基，并用0.5ml PBSTx（磷酸盐缓冲盐水，PBS，用0.3％的Triton X-100），一次冲洗的大脑。让大脑沉淀到的所有液体之间交流的试管底部。的曲拉通X-100洗涤剂，用于透化组织。清洗通常需要不到30秒;只需删除PBSTx一旦大脑纷纷落户到管底。 </li><li>用0.5毫升的9％电镜观察级的多聚甲醛稀释于PBSTx更换溶液。孵育章动15分钟，在室温下进行。 </li><li>根据危险废物处置条例固定液。 </li><li>洗样品用0.5ml PBSTx 15分钟。重复该洗涤步骤用新鲜PBSTx两次以上。 </li><li>用0.5毫升的PBT（PBS，1更换解决方案％牛血清白蛋白（BSA）和0.1％吐温20）。孵育垂头为1小时，在室温下进行。这一步块抗体与组织中的非特异性结合。 </li><li>用稀释在PBT中添加4％正常山羊血清（NGS）0.5毫升初级抗体替代的解决方案。与章动孵育过夜，4°C。 </li><li>丢弃或保存稀释的一抗。 </li><li>洗样品用0.5ml的PBT 15分钟。重复该洗涤步骤用新鲜的PBT两次以上。 </li><li>用0.5ml改性的PBT（PBS，2％BSA，0.1％Tween-20中，和4％NGS）更换溶液。孵育垂头为1小时，在室温下进行。 </li><li>用稀释成改性PBT0.5毫升二抗更换解决方案。孵育垂头为2小时，在室温下进行。 </li><li>用0.5ml PBST（PBS，0.1％吐温20）代替溶液。孵育章动15分钟，在室温下进行。重复该洗涤步骤用新鲜的PBST两次以上。 </li></ol><ol><li>小心转移样品为在载玻片上脱落，限制对滑动的中心左侧的下降。 </li><li>加安装介质的大颗粒（直径约2厘米）（例如，Aqua-Poly/Mount）以滑动的中心。避免PBST的是朝左的游泳池。 </li><li>使用镊子的大脑从PBST池转移到安装介质池。直接在大脑的顶部添加安装介质可能会破坏它们的方向，应该避免。 </li><li>在光学显微镜下，安排样品中的安装介质中，将成像的一面朝上。 </li><li>在光学显微镜下滑动，慢慢降低对样品的顶部＃1.5盖玻片。可选步骤:通过在盖玻片轻轻推打跑小的气泡。 </li><li>允许安装介质进行聚合数小时，或过夜，以躺着的幻灯片平坦，光保护，在室温下进行。 </li><li>幻灯片可在第二天成像，或之前成像存储。 </li></ol>

<ol>
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H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Siegrist, S. E., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extrinsic+cues+orient+the+cell+division+axis+in+Drosophila+embryonic+neuroblasts.">Extrinsic cues orient the cell division axis in Drosophila embryonic neuroblasts.</a> Development. 133, 529-536 (2006).</li><li>Broadus, J., Doe, C. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+pericentrin-like+protein+is+essential+for+cilia/flagella+function,+but+appears+to+be+dispensable+for+mitosis.">The Drosophila pericentrin-like protein is essential for cilia/flagella function, but appears to be dispensable for mitosis.</a> J. Cell Biol. 165, 673-683 (2004).</li><li>Dix, C. I., Raff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Spd-2+recruits+PCM+to+the+sperm+centriole,+but+is+dispensable+for+centriole+duplication.">Drosophila Spd-2 recruits PCM to the sperm centriole, but is dispensable for centriole duplication.</a> Curr. Biol. 17, 1759-1764 (2007).</li><li>Moutinho-Santos, T., Sampaio, P., Amorim, I., Costa, M., Sunkel, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+localisation+of+the+mitotic+POLO+kinase+shows+a+highly+dynamic+association+with+the+mitotic+apparatus+during+early+embryogenesis+in+Drosophila.">In vivo localisation of the mitotic POLO kinase shows a highly dynamic association with the mitotic apparatus during early embryogenesis in Drosophila.</a> Biol. Cell. 91, 585-596 (1999).</li><li>Megraw, T. L., Kilaru, S., Turner, F. R., Kaufman, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+centrosome+is+a+dynamic+structure+that+ejects+PCM+flares.">The centrosome is a dynamic structure that ejects PCM flares.</a> J. Cell Sci. 115, 4707-4718 (2002).</li></ol>

作者什么都没有透露。

活细胞成像是用来研究，规范ACD干细胞，细胞谱系的分化机制的宝贵方法，以及复杂的组织形态。虽然研究组历来采用了各种技术来可视化NB ACD，这些方法可大致分为两类即主要与脑组织是否是原封不动或解离的差异。 成像分离的NB有其优势。为1，解离的NB更易图像，因为它们是特别容易识别作为最大的（直径约12微米）的细胞在培养皿。此外，几乎所有的干细胞，培养在玻?...

干细胞保持分化的平衡和自我更新的早期发展过程中产生细胞多样性和替代受损的细胞在成体组织。这种稳态的调节可以防止两者的损耗和过度膨胀的干细胞群体，这可能会导致有害的组织变性或肿瘤的发生。 神经母细胞（NBS）是果蝇中枢神经系统（ 图1A）在胚胎阶段1，2是第一种形式的广泛研究的神经干细胞。 NB的经过反复多轮的不对称细胞分裂（ACD），产生不等价的两个注定细胞:一个自我更新的干细胞和分化细胞。 ACD是由中心体，充当大多数细胞3的微管组织中心的非膜性细胞器引导。在有丝分裂，NB中心体组织和定向双极纺锤体沿顶 - 基底极性性轴。待分割NB的乳沟，根尖命运决定了指定干细胞的命运，和基底命运决定指定分化，被分隔成不平等的子细胞。 在幼虫中央脑，两种类型的NB可以通过它们的数量，位置，转录因子的表达，细胞谱系（ 图1A）4-6相区别。 I型的NB是最丰富的，并且其中约90填充大脑7的各视叶的前部和后部两侧。这些NB的表达的转录因子Asense（ASE），并且它们特征性地划分成一个自我更新的NB和1小神经节母细胞（GMC; 图1B）。每个GMC经历了一个单一的终端部门产生两个神经元或神经胶质细胞（ 图1B）。与此相反，8型II的NB是填充每个视叶的后侧缺乏日月光表达式5。他们经过ACD要生成一个自我更新的NB和一个中间神经祖（INP）。 该INP，反过来，划分不对称的三到五倍。所有这些分歧导致了INP的再生和生产单GMC 4。统，具体的NB身份和GMC出生的时间次序引起的成人中枢神经系统的神经细胞的惊人的多样性。 了解细胞生物学的基础是NB ACD已通过使用活细胞成像技术大大提高。研究人员使用到的图像实时的NB发布的协议有很大的不同。但是，总的来说，这些方法可以被分成由幼虫大脑是否保持不变或机械分离区分两种主要类别。这两种方法具有不同的优点和缺点取决于研究者的应用。 早期的报告揭示活NB细胞分裂本期开始涉及一定程度的幼虫大脑手动解离。这些协议的细节涂抹8或戏弄9外脑，以发展国家统计局对玻璃盖玻片短期原代培养。以改善成像，该圆的NB通常是扁平的上与任一载玻片8或琼脂糖垫9的盖玻片。虽然扁平的细胞具有改进的光学系统，这些技术往往导致NB的有丝分裂缺陷，包括卵裂沟的回归和无法分裂一次以上。因此，包含手工解离和幼虫脑组织的物理畸变的协议一般只适用于非常短期的（ 即 ，，一个细胞周期或更少）的应用程序。同样地，半的挤压或完全压扁玻璃载玻片和盖玻片之间完整脑限制一个时间可能会花费成像一个给定的样本，以一个约30分钟的时间内10。尽管有此项限制，氏的方法已经成功地11-14使用。 最近努力图像实时解离的NB极限的分离细胞的物理变形。这些技术的应用中，有必要维持细胞培养物中对多个细胞分裂周期中特别有用。例如，分散的细胞从手动分离大脑可以部分地埋入在由纤维蛋白原和凝血酶15长期成像16的混合物制成的凝块。可替代地，外植大脑可能是第一化学解离与酶胶原酶，通过移液管尖下一个手动打乱重复通道，并且随后接种于聚-L-赖氨酸包被的玻璃底培养皿17，18。镀膜玻璃的菜肴与任何纤维蛋白原或聚-L-赖氨酸促进附件和轻微的圆形细胞的扩散，使它们尽可能靠近盖玻片尽量不重大的物理变形。在additi上，这些方法涉及可方便地更换药理扰动实验18培养基中培养国家统计局。总体而言，直接电镀分散的NB改善成像光学元件在不牺牲长期的成像能力。近日，游离描述国家统计局的长期培养的协议已经有详细19。 然而，这种方法并非没有其局限性。有几个注意事项，以实时成像分离的NB。在分散的细胞的一个字段，例如，它是很难识别在空间组织中的完整的脑1特定NB谱系。同样，区分类型I与类型分离的组织内II的NB也是具有挑战性的无谱系示踪物或亚型特异性转基因，如worniu-GAL4，asense-GAL80 20的表达。虽然这些转基因是在一些应用中非常有用，它们限制了研究人员对这些转基因前在UAS增强子元件21的控制下，按下并需要更复杂的遗传计划。的研究，涉及的NB的空间或时间的发展，因此，需要在一个完整的组织的情况下体内成像。 此外，分离的胚胎的NB的研究表明，对于关键的极性相间的定位决定因素22，23相邻的小区的物理接触是至关重要的。这些研究表明完全隔离的NB不再保持不变有丝分裂纺锤体轴。相反，这些细胞显示出更多的随机主轴和连续GMC芽之间的分离，宽角度或许是由于根尖定位中心体22的损失。虽然幼虫国家统计局和他们相邻小区之间的关系还没有被广泛研究，这是值得考虑的幼虫干细胞微环境可能扑来细胞极性或其他行为。为了保持幼虫的NB的生理情况下，我们形象的ACD从整个大脑。 鉴于与成像分离的NB相关的固有局限性，我们的实验室和其他人建立了协议，从整个幼虫的大脑图像连续两轮NB的ACD。技术，以图像完整脑常与柔性膜，以防止组织变形或损坏，并允许气体交换取代玻璃的刚性表面上的培养容器的一侧。一些方法涉及装配取出的大脑中的昆虫培养用培养基，血清和抗坏血酸与10幼虫24中分离的脂肪体的混合物。分离出的脂肪组织被添加，因为它们分泌可以刺激NB细胞分裂25有丝分裂原。这个协议保留组织的完整性超过3小时，因此，适合于长期成像24，26-28。近日，描述LON协议在抗坏血酸，血清和脂肪组织的存在完好幼虫大脑的克长期成像已详述29。重要的是，因为该组织既不暴露也不固定，这种方式是为应用而培养介质被交换的有用（ 例如 ，药物研究，免疫耗竭， 等等 ）。 我们的实验室简化了整个安装现场准备幼虫大脑长期成像30，31。主要的优点我们的通讯协议在别人就是它的简单:我们的观察表明既不血清，抗坏血酸，胰岛素，也不胖的身体是必要的添加剂，以图像连续两轮NB的ACD。虽然添加剂，如胰岛素，已可用于干细胞分裂32和果蝇卵巢33集体细胞迁移的长时间成像，它们似乎是可有可无的NB ACD，作为我们的成像介质包括STA的简单混合物的ndard昆虫细胞培养用培养基补充有抗生素 - 抗真菌剂以防止污染。通过使用可重复使用的透气性或玻璃底培养皿中，我们已经能够维持几轮连续NB的ACD的。相同的外植样品可以容易地被用于免疫和其他程序与固定的组织。在这个协议中，我们详细介绍完整的幼虫大脑的解剖，以及编制大脑的实时和固定的分析。

<table border="0" cellpadding="0" cellspacing="0" width="851">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Lumox Tissue Culture Dish 50X12 mm</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:119px;">94.6077.410</td>
			<td style="width:167px;">A reusable culture dish with a gas-permeable and optically clear film base.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Schneider&#39;s S2 Medium</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:119px;">21720-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Gibco Antibiotic-Antimycotic (100x)</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:119px;">15240-062</td>
			<td>A supplement containing penicillin (10,000 U/mL), streptomycin (10,000 mg/mL), and amphotericin B (25 mg/mL) that is added to Schneider&#39;s S2 media to prevent bacterial and fungal contamination.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Glass coverslips, #1.5 22X22 mm</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:119px;">12-541-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Halocarbon oil 700</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:119px;">H8898</td>
			<td>A high viscosity inert oil that is clear and colorless.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">pair of nickel-plated pin holders, 17 cm long</td>
			<td style="width:185px;">Fine Science Tools</td>
			<td style="width:119px;">26018-17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Minutien dissecting pins</td>
			<td style="width:185px;">Fine Science Tools</td>
			<td style="width:119px;">26002-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">pair of Dumont #5 Forceps</td>
			<td style="width:185px;">Fine Science Tools</td>
			<td style="width:119px;">11252-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Dissecting needle/probe with plastic handle</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:119px;">08-965-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Syringe filter, 0.22 mm pore size</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:119px;">09-719A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Syringe 5 mL&#160;</td>
			<td style="width:185px;">BD Biosciences</td>
			<td style="width:119px;">309646</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">plastic transfer pipet</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:119px;">S30467-1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:380px;">paraformaldehyde, 32% EM-grade</td>
			<td style="width:185px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15714</td>
			<td>CAUTION: Formaldehyde is toxic; it should be handled in a fume hood. Follow MSDS guidelines for safe handling.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">DAPI (4&#39;, 6-diamidino-2-phenylindole, dihydrochloride)</td>
			<td style="width:185px;">Invitrogen</td>
			<td style="width:119px;">D1306</td>
			<td>A dye that labels DNA and is excited by UV light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Aqua/PolyMount; Polysciences, Inc.</td>
			<td style="width:185px;">Fisher Scientific</td>
			<td style="width:119px;">18606-20</td>
			<td>A water-soluble mounting medium.</td>
		</tr>
	</tbody>
</table>

live imaging of drosophila larval neuroblasts

干细胞不对称分裂产生两个子代细胞与不平等的命运潜能:是一个自我更新的干细胞和分化细胞。鉴于其相关性发育和疾病，了解支配不对称的干细胞分裂一直是研究的一个强大的区域机制。因为他们是基因听话，并进行连续几轮细胞分裂约每小时一次， 果蝇中枢神经系统，或神经母细胞的干细胞，是不可缺少的型号为干细胞分裂的研究。约100个​​神经干细胞是位于每两个幼虫脑叶的表面附近，使该模型系统，用于实时成像显微镜研究特别有用。在这项工作中，我们将回顾一些方法广泛用于可视化的干细胞分裂，和我们处理的相对优势和那些采用分离与完整的大脑组织中的技术缺点。我们还详细介绍了simplifIED协议，用于从三龄幼虫活细胞成像和固定分析应用外植整个大脑。

实时成像已经阐明了多项调控NB的亚洲合作对话机制。前图像的采集，它是必要的，以确定最佳成像条件。有必要与活细胞成像时间平衡帧捕获率。对细胞的分析，例如，增加的时间和可能需要的光学分辨率。当可视化单一NB细胞周期（ 图4A），在该图像被捕获的速率可以增加，而不会引入光损伤。通常情况下，单个细胞分裂事件可以在约10-30秒的时间间隔进行监测。以提高时间分辨?...

Watch this Scientific Journal Video about Live Imaging of Drosophila Larval Neuroblasts at JoVE.com

Live Imaging of Drosophila Larval Neuroblasts

该协议详细说明了用于进行活细胞成像在一个完整的幼体脑的情况下简化方法。活细胞成像的方法是无价的，不对称的神经干细胞分裂的研究以及其他神经和发育过程，坚持揭露先前忽略的机制。

的实时成像<em&gt;果蝇</em&gt;幼虫神经母细胞

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

live-imaging-of-drosophila-larval-neuroblasts

Research

JoVE Journal

Neuroscience

Live Imaging of Drosophila Larval Neuroblasts

14.7K 次观看.  National Institutes of Health. 该协议详细说明了用于进行活细胞成像在一个完整的幼体脑的情况下简化方法。活细胞成像的方法是无价的，不对称的神经干细胞分裂的研究以及其他神经和发育过程，坚持揭露先前忽略的机制。 

视频: Live Imaging of Drosophila Larval Neuroblasts

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

High-Resolution Magic Angle Spinning (HRMAS) proton magnetic resonance spectroscopy (1H-MRS) is a novel non-destructive technique that improves spectral line-widths and allows high-resolution spectra to be obtained from extracts, intact cells, cell cultures, and more importantly intact tissue to investigate relationships between metabolites and cellular processes. In vivo HRMAS 1H-MRS studies have yet to be reported in the live fruit fly Drosophila melanogaster. Drosophila, as a simpler genetic organism, allows the multiple biological functions and various evolutionarily conserved signaling pathways to be examined at the whole organism level and it is a useful model for investigating genetics and physiology. To this end, we developed and implemented an in vivo HRMAS 1H-MRS method to investigate live Drosophila at 14.1 T. Here, we outline an HRMAS 1H-MRS protocol for the molecular characterization of Drosophila with a conventional MR spectrometer equipped with an HRMAS probe. This technique is a novel, in vivo, non-destructive Drosophila metabolite measurement approach, which enables the identification of disease biomarkers and thus may contribute to novel therapeutic development.

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

This technique enables the use of high-resolution magic angle spinning proton MR spectroscopy (HRMAS 1H-MRS) for molecular characterization of live Drosophila melanogaster with a conventional 14.1 tesla spectrometer equipped with an HRMAS probe.

生活磁共振波谱果蝇</em用魔角旋转

High-Resolution Magic Angle Spinning (HRMAS) proton magnetic resonance spectroscopy (1H-MRS) is a novel non-destructive technique that improves spectral ...

科研

神经科学

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

3幼虫节段性神经的可视化 RD龄果蝇幼虫的筹备工作

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

在果蝇幼虫分部轴突在体内的突触小泡的可视化

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Live Imaging of Dorsal Root Axons after Rhizotomy

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. Regeneration of dorsal root (DR) axons into spinal cord is prevented at the dorsal root entry zone (DREZ), the interface between the CNS and PNS. Our understanding of the molecular and cellular events that prevent regeneration at DREZ is incomplete, in part because complex changes associated with nerve injury have been deduced from postmortem analyses. Dynamic cellular processes, such as axon regeneration, are best studied with techniques that capture real-time events with multiple observations of each living animal. Our ability to monitor neurons serially in vivo has increased dramatically owing to revolutionary innovations in optics and mouse transgenics. Several lines of thy1-GFP transgenic mice, in which subsets of neurons are genetically labeled in distinct fluorescent colors, permit individual neurons to be imaged in vivo1. These mice have been used extensively for in vivo imaging of muscle2-4 and brain5-7, and have provided novel insights into physiological mechanisms that static analyses could not have resolved. Imaging studies of neurons in living spinal cord have only recently begun. Lichtman and his colleagues first demonstrated their feasibility by tracking injured dorsal column (DC) axons with wide-field microscopy8,9. Multi-photon in vivo imaging of deeply positioned DC axons, microglia and blood vessels has also been accomplished10. Over the last few years, we have pioneered in applying in vivo imaging to monitor regeneration of DR axons using wide-field microscopy and H line of thy1-YFP mice. These studies have led us to a novel hypothesis about why DR axons are prevented from regenerating within the spinal cord11.
In H line of thy1-YFP mice, distinct YFP+ axons are superficially positioned, which allows several axons to be monitored simultaneously. We have learned that DR axons arriving at DREZ are better imaged in lumbar than in cervical spinal cord. In the present report we describe several strategies that we have found useful to assure successful long-term and repeated imaging of regenerating DR axons. These include methods that eliminate repeated intubation and respiratory interruption, minimize surgery-associated stress and scar formation, and acquire stable images at high resolution without phototoxicity.

An in vivo imaging protocol to monitor primary sensory axons following dorsal root crush is described. The procedures utilize wide-field fluorescence microscopy and thy1-YFP transgenic mice, and permit repeated imaging of axon regeneration over 4 cm in the PNS and axon interactions with the interface of the CNS.

实时成像背根轴突后根切断术

The primary sensory axons injured by spinal root injuries fail to regenerate into the spinal cord, leading to chronic pain and permanent sensory loss. ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

幼虫，蛹和成人的解剖和免疫组化<em&gt;果蝇</em&gt;视网膜

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

在幼虫馈电电路的功能分析果蝇</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

为什么量化事项:表型的表征在<em&gt;果蝇</em&gt;幼虫神经肌肉接头

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

用线性琼脂糖渠道研究<em&gt;果蝇</em&gt;幼虫爬行行为

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

从果蝇幼虫神经肌肉交界处的突触电流的焦 Macropatch 记录

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...