我们非常感谢埃里克Wieschaus，提供了该协议开发的基础上。我们的工作是一个贝尔纳Marrs麦克莱恩部生物化学与分子生物学启动奖，贝勒医学院的支持。

1。装配胚胎收集杯<ol><li>切底关闭100 mL的烧杯用剃刀三玉米，尽可能顺利的边缘。杯子很容易处理，如果你还修剪关闭顶部的三个角，虽然这不是绝对必要的。 </li><li>切丝网（6厘米x 6厘米）的平方。在一个预热的热点板块，里面的通风橱，层丝网一块重型铝箔上的平方。推杯的削减，坚决到热网的底部边缘。等待几秒钟，并解除与现在连接的网状杯。如果箔也坚持，只是剥离其关闭。 </li><li>酷杯过夜。关闭任何尖锐的细粒度砂纸边缘，删除多余的网，用剪刀和沙子。 </li></ol> 2。让苹果汁琼脂平板<ol><li>在6L烧瓶中，结合100克BD细菌用琼脂和3L蒸馏水。高压灭菌30分钟缓慢排气设置的琼脂。 </li><li>在一个搅拌棒2L容量瓶中，结合100克蔗糖，苹果汁1L和6克对羟基苯甲酸。热到沸腾，而上一个热点板块搅拌的解决方案。不要煮沸2分钟以上。允许苹果汁混合，冷却，然后添加琼脂用搅拌棒。混合完全。 </li><li>允许合并的解决方案，在60℃水浴冷却，然后再浇筑到60x15 mm培养皿中。另外，蠕动泵可用于免除琼脂。允许板冷却到室温，至少4个小时。 Rubbermaid的容器，在4 ° C的湿纸巾和存储层的堆栈</li></ol>注: <ul><li>在秋季和冬季，可能有必要增加一个额外的100毫升的水琼脂。 </li><li>皮氏培养皿中的品牌是很重要的！只有屋宇署猎鹰菜适合三玉米烧杯。具体的订购​​信息中可以找到下面的材料表。 </li></ul> 3。将GFP的苍蝇胚胎收集杯<ol><li> GFP的股票，根据到您的收藏需求展开设立瓶苍蝇大约两个星期前成像。苍蝇一瓶通常是绰绰有余的，以填补与最低的50位女性和30男性胚胎收集杯。最好的铺设，苍蝇应新eclosed（即少于5天的孵化后）。 </li><li>请填写大致相等的部分红星积极干燥酵母和蒸馏水一小杯粘贴酵母，搅拌直至溶解酵母。粘贴应近似湿花生酱的一致性。可以添加更多的酵母或水改变的一致性。酵母膏，可使用数天，应贮存，覆盖，在4 ° C。 </li><li>酵母膏一旦准备就绪，删除苹果汁板从4 ° C储存。数的苹果汁，酵母膏板，并允许他们温暖，以22-25 ° C，这将鼓励产蛋。 </li><li>圆形滤纸折一半，再一半。修剪直径8-9厘米，并在四分之一圆的手​​风琴褶皱。开展了一圈，倒置，插入一个收集杯，直到它触及网格。确保纸张在世界杯的安全，以便它不属于粉碎苍蝇。滤纸是可选的，但确实提供了一个温馨的环境进行交配，并保持湿度杯。 </li><li>标签一杯股票的名称和日期。苍蝇从瓶子转移到收集杯轻轻晃动在杯子倒瓶，并立即用准备好的苹果汁板盖杯。一杯用橡皮筋固定板。设置杯网状侧面积直射光。确保没有阴影落在杯中，苍蝇不会在黑暗中奠定。 </li><li>需要更改苹果汁板。两小时的集合，在室温下，成像cellularization。 </li></ol> 4。准备安装室<ol><li>要准备一个胚胎的安装室，切2-3厘米长的一块双面胶带和它放在幻灯片上，对准磁带和幻灯片的长轴。切2-3厘米长的一块双面胶带层的其他磁带上，确保他们的边缘对齐。 </li><li>使用刀片，使两个中心的磁带，并垂直于长轴的幻灯片削减约3毫米。删除磁带之间的削减，使一个通道。吸取一个下降通道的卤烃27油。此通道的胚胎将被安装。 </li></ol>注: <ul><li>只有0.5英寸苏格兰双面胶带，是正确的厚度，以适应胚胎。 </li><li> 27卤烃油透氧，使氧气的交换，同时防止胚胎脱水。由于它的折射率，卤烃27石油也是理想的分期成像胚胎。 </li></ul> 5。 Dechorionate胚胎<ol><li>要收集和dechorionate胚胎，倒入足够的苹果汁琼脂平板上，以完全淹没整个表面的50％漂白粉。如蔡司发现V8透射光，使用解剖显微镜，观察胚胎释放他们的绒毛膜。只要绒毛膜放松，从一个胚胎数（30-60秒）发布，倒入一个细胞过滤器的漂白剂和胚胎。 </li><li>从喷瓶蒸馏水洗净滤网胚胎立即大力。民建联过滤纸巾。如果任何粉红色毛巾涂抹后，继续用清水冲洗胚胎。 </li><li>使用湿画笔10-50胚胎转移到一个干净的苹果汁板（无酵母膏）。威克远离纸巾撕边的水，并立即支付少量的卤烃27油胚胎。 </li></ol>注: <ul><li>不要使用高乐氏漂白。相反，使用一个品牌，作为奥斯汀的A - 1商业漂白剂，这是&lt;6％次氯酸钠等。 </li><li>过度漂白或不足冲洗会导致不规则cellularization糊状胚胎。 </li></ul> 6。舞台及摩胚胎<ol><li>使用透射光的夹层的显微镜，按照Bownes 20的形态指引到上盘阶段的胚胎。使用镊子，转让5有丝分裂周期11或12个胚胎，对应到Bownes第4阶段，安装室的通道。排列在一条线上的胚胎，背，腹两侧可见的和横向的一面朝下。胚胎很容易在这仅在石油的方向排列。不要人群胚胎，因为他们的氧气会剥夺他们的邻居一次盖玻片应用于如下。 （保持它们之间的胚胎长度约一半）。 </li><li>打好一个25X25毫米盖的一个边缘滑双面胶带在通道的一侧。除去盖玻片盖通道。如果是抓下面的空气，适用于盖玻片边缘的卤烃27油少量。的毛细作用，将拉进油的渠道，推动了空气。 </li></ol>注: <ul><li>举办​​胚胎的另一个优秀资源如下:坎波斯，奥尔特加，JA和Hartenstein，五（1985年）。 果蝇的胚胎发育。斯普林格出版社，柏林。虽然这本书不再出版，使用副本定期对Amazon.com。 </li><li>务必谨慎使用的卤烃27油。这将使得安装更加容易。它也将防止意外显微镜物镜的油渗出到以下的成像步骤。 </li></ul> 7。图片胚胎<ol><li>如何你继续在这里当然会取决于细胞形状的变化，你是成像，和解决的问题，你。任何形状的变化，您的安装室放在一个直立或倒置显微镜，透射光找到你的胚胎，然后切换到共焦成像，把重点放在地区的利益。坚持的最佳做法，如共聚焦显微镜， <a href="http://www.microscopyu.com/articles/confocal">http://www.microscopyu.com/articles/confocal</a>了尼康的MicroscopyU（讨论）。特别是在揭露你的活胚胎，以尽可能少的激光功率，尽可能严谨，以防止光漂白和光毒性。 </li><li> cellularization，我们蔡司710激光扫描共聚焦显微镜图像，使用了C -复消色差透镜40x/1.2 W更正 ​​目标。这种显微镜被安置在房间温度控制，温度范围从18-20 ° C。虽然温度控制也不是绝对必要的，这是为更好的显微镜，使成像更一致的。我们经常在一个单一的平面图像，附近的胚胎中，按照截面的质膜内陷（图2）。以下内陷动态，有30-60秒的时间间隔足够的时间推移成像，胚胎应能承受90分钟成像缺氧没有明显的迹象。 </li><li>获取图像后，他们可以分析使用任何图像分析软件包，包括从美国国立卫生研究院，免费ImageJ（ <a href="http://rsbweb.nih.gov/ij">http://rsbweb.nih.gov/ij</a> ）。的数据，然后可以作为电影（电影），序列（图2），或kymographs 21 。 </li></ol>注意: <ul><li>浸水的C复消色差透镜40x/1.2 W更正 ​​目标是附近的胚胎中间部分是由于其高的光圈，这限制了水样中的深部组织成像畸变的成像非常适合，并给出了高分辨率和高荧光信号。此外，这个目标有一个很长的工作距离（220微米）。然而，其他的水或甘油中浸泡目标将表现良好，特别是如果感兴趣的细胞形状的变化可以成像胚胎表面附近。 </li></ul> 8。另一种方法:"胚胎胶"山胚胎<ol><li>图像细胞形状改变比cellularization，它可能是需要安装在一个特定的方向，是不容易维持在石油的胚胎。要做到这一点，使用一个备用的安装方法前面介绍的。相结合，与250μL的正庚烷在闪烁瓶中的20厘米的双面胶带"胚胎胶"开始。广场上nutator的闪烁瓶或旋转平台，混合过夜。 </li><li>胚胎胶浸入黄色的枪头，然后跟踪沿幻灯片的一角，留下的胶水的痕迹。虽然庚烷是蒸发，对准你的上演在所需方向上的琼脂块的胚胎。 （请记住，胚胎表面进行成像，应面临的琼脂。形象在腹沟形成的细胞形状的变化，例如，安装与他们的腹侧方面临的琼脂胚胎。） </li><li>反转用胶水的幻灯片，并轻轻按压对胚胎的琼脂块上的胶水的痕迹。现在再次反转幻灯片。胚胎将停留在正确的方向。添加胚胎两侧的两层双面胶，盖卤烃27油，适用于盖玻片。继续与上文所述的成像。 </li></ol> 9。代表性的成果: 如果胚胎是健康的，成像是最优的，那么应该采取cellularization 50-60分钟，质膜内陷入口近40微米。然而，如果胚胎是过度漂白，氧，光毒性剥夺或损坏，然后内陷将缓慢或停止，尤其是在成像领域。这种胚胎健康恶化，结果往往是改变发展，幼虫孵化失败。因此，严格的测试后成像技术来检测胚胎健康，在湿盒中保持你的幻灯片，并观看孵化第二天。 <img src="/files/ftp_upload/2503/2503fig1.jpg" alt="图1" /> 图1。从胚胎收集成像的工作流程，该协议的工作流程可以分成四个主要阶段。在第一阶段，所有的个人用品和组件准备，胚胎收集杯，苹果汁琼脂平板和GFP果蝇放在一起创建的胚胎铺设的环境。在第二阶段中收集，鸡蛋和苹果汁琼脂平板上奠定了胚胎。在第三阶段，胚胎是从板块中删除，上演，并转移到装配室。在第四阶段，安装胚胎共聚焦显微镜成像。 <img src="/files/ftp_upload/2503/2503fig2.jpg" alt="图2" /> 图2。安装有代表性的数据，从时间推移cellularization成像胚胎背侧（D）和腹（五）双方清晰可见，和附近的中间影像，按照截面质膜内陷。这里显示的胚胎表达了绿色荧光蛋白-肌球蛋白- 2探头6，集中在质膜内陷提示。因此，跟踪这个随着时间的推移前面的侵入，使invaginates质膜的速率。 0:00分钟的时间点对应cellularization发病。 56:00分钟的时间点后不久，原肠开始对胚胎的腹侧方。酒吧是40微米。 电影1。从时间推移cellularization成像代表电影这部电影。对应于图2。为了记录整个cellularization过程，成像在事先有丝分裂周期13的开始，捕捉pseudocleavage畦回归，并继续对胚胎的腹侧方，直到原肠运动。图片收集在一分钟的时间间隔。强度增加收购后，使其更容易看到的原肠运动。 <a href="http://www.jove.com/files/ftp_upload/2503/Movie%201.mov">点击这里为电影</a> <table border="1"><tbody><tr><td> 发展事件和时序* </td><td> 相关的细胞形态变化</td><td> 疾病或人体健康的链接</td><td> 最近提及实时成像 </td></tr><tr><td>伪分裂沟的形成 （4; 90分钟PF） </td><td>胞质</td><td>多倍体和癌症的发展1 </td><td> Mavrakis等，2009年8 曹等人，2010年9 </td></tr><tr><td> Cellularization （5; 130分钟PF） </td><td>胞质</td><td>多倍体和癌症的发展1 </td><td>曹等人，2008年10月 Sokac Wieschaus，2008年11月 </td></tr><tr><td>腹畦形成，中胚层内陷 （6; 180分钟PF） </td><td>心尖​​收缩;上皮 - 间质转型</td><td>癌转移2 </td><td>福克斯Peifer，2007年12月 马丁等人，2009年13 </td></tr><tr><td> Germband扩展 （第7条; 195分钟PF） </td><td>收敛延伸</td><td>神经管缺陷3 </td><td> Bertet等，2004年第14 布兰肯希普等人，2006年15 </td></tr><tr><td> Tracheogenesis （11; 320分钟PF） </td><td>上皮管形成和分支</td><td>血管生成4 </td><td> Caussinus等，2008 16 热尔韦与卡萨诺瓦，2010 17 </td></tr><tr><td>背封 （14; 620分钟PF） </td><td>心尖​​收缩</td><td>伤口愈合5 </td><td> Gorfinkiel等，2009年第18 梭伦等人，2009 19 </td></tr></tbody></table>例子成像在生活飞胚胎细胞形态的变化见表1 。 Bownes阶段的数量和时间受精后（PF），每个事件开始时，根据坎波斯，奥尔特加，1985年上市。 <table border="1"><tbody><tr><td> 飞股票 </td><td> 标签 </td><td> 原始参考 </td></tr><tr><td>蜘蛛GFP（95-1） </td><td>质膜</td><td>莫兰等人，2001年22 </td></tr><tr><td> Resille - GFP（117-2） </td><td>质膜</td><td>莫兰等人，2001年22 </td></tr><tr><td> GAP43金星</td><td>质膜</td><td> Mavrakis等，2009年8 </td></tr><tr><td>意大利面条壁球- GFP（Sqh - GFP） </td><td>肌球蛋白2 </td><td> Royou等，2002年6 </td></tr><tr><td> E - cadherin的GFP（ECAD - GFP） </td><td>细胞与细胞结</td><td>小田等，2001年23 </td></tr><tr><td> GFP - Moesin </td><td> F -肌动蛋白</td><td> Kiehart等，2000年第24 </td></tr><tr><td> Utrophin金星（Utro金星） </td><td> F -肌动蛋白</td><td> Sokac等，未公布结果</td></tr></tbody></table>有用的股票为成像飞胚胎细胞形状的变化见表2。

<ol>
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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+contractions+of+an+actin-myosin+network+drive+apical+constriction.">Pulsed contractions of an actin-myosin network drive apical constriction.</a> Nature. 457, 495-499 (2009).</li><li>Bertet, C., Sulak, L., Lecuit, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin-dependent+junction+remodelling+controls+planar+cell+intercalation+and+axis+elongation.">Myosin-dependent junction remodelling controls planar cell intercalation and axis elongation.</a> Nature. 429, 667-671 (2004).</li><li>Blankenship, J. T., Backovic, S. T., Sanny, J. S., Weitz, O., Zallen, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+rosette+formation+links+planar+cell+polarity+to+tissue+morphogenesis.">Multicellular rosette formation links planar cell polarity to tissue morphogenesis.</a> Dev Cell. 11, 459-470 (2006).</li><li>Caussinus, E., Colombelli, J., Affolter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tip-cell+migration+controls+stalk-cell+intercalation+during+Drosophila+tracheal+tube+elongation.">Tip-cell migration controls stalk-cell intercalation during Drosophila tracheal tube elongation.</a> Curr Biol. 18, 1727-1734 (2008).</li><li>Gervais, L., Casanova, J. <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+coupling+of+cell+elongation+and+lumen+formation+in+a+single+cell.">In vivo coupling of cell elongation and lumen formation in a single cell.</a> Curr Biol. 20, 359-366 (2010).</li><li>Gorfinkiel, N., Blanchard, G. B., Adams, R. J., Arias, M. a. r. t. i. n. e. z., A, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+control+of+global+cell+behaviour+during+dorsal+closure+in+Drosophila.">Mechanical control of global cell behaviour during dorsal closure in Drosophila.</a> Development. 136, 1889-1898 (2009).</li><li>Solon, J., Kaya-Copur, A., Colombelli, J., Brunner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulsed+forces+timed+by+a+ratchet-like+mechanism+drive+directed+tissue+movement+during+dorsal+closure.">Pulsed forces timed by a ratchet-like mechanism drive directed tissue movement during dorsal closure.</a> Cell. 137, 1331-1342 (2009).</li><li>Bownes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photographic+study+of+development+in+the+living+embryo+of+Drosophila+melanogaster.">A photographic study of development in the living embryo of Drosophila melanogaster.</a> J Embryol Exp Morphol. 33, 789-801 (1975).</li><li>Sokac, A. M., Wieschaus, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zygotically+controlled+F-actin+establishes+cortical+compartments+to+stabilize+furrows+during+Drosophila+cellularization.">Zygotically controlled F-actin establishes cortical compartments to stabilize furrows during Drosophila cellularization.</a> J Cell Sci. 121, 1815-1824 (2008).</li><li>Morin, X., Daneman, R., Zavortink, M., Chia, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protein+trap+strategy+to+detect+GFP-tagged+proteins+expressed+from+their+endogenous+loci+in+Drosophila.">A protein trap strategy to detect GFP-tagged proteins expressed from their endogenous loci in Drosophila.</a> Proc Natl Acad Sci U S A. 98, 15050-15055 (2001).</li><li>Oda, H., Tsukita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+cell-cell+adherens+junctions+reveals+that+Drosophila+mesoderm+invagination+begins+with+two+phases+of+apical+constriction+of+cells.">Real-time imaging of cell-cell adherens junctions reveals that Drosophila mesoderm invagination begins with two phases of apical constriction of cells.</a> J Cell Sci. 114, 493-501 (2001).</li><li>Kiehart, D. P., Galbraith, C. G., Edwards, K. A., Rickoll, W. L., Montague, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+forces+contribute+to+cell+sheet+morphogenesis+for+dorsal+closure+in+Drosophila.">Multiple forces contribute to cell sheet morphogenesis for dorsal closure in Drosophila.</a> J Cell Biol. 149, 471-490 (2000).</li></ol>

本文所述的协议将允许生活，在发展中国家果蝇胚胎的细胞形态变化的共焦成像。成像GFP的股票，可以由个人实验室（见表2）准备，但许多这类股票也公开如布卢明顿果蝇联合中心的中心在美国印第安纳大学<a href="http://flystocks.bio.indiana.edu">（</a> http://flystocks.bio.indiana.edu）和捕蝇草在美国耶鲁大学（ <a href="http://flytrap.med.yale.edu">http://flytrap.med.yale.edu</a> ）联合中心。一旦获得这些实时成像?...

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imaging cell shape change in living drosophila embryos

发展的果蝇胚胎发生细胞形态的变化，非常适合现场共焦成像。在果蝇的细胞形状的变化类似于在高等生物的，他们驾驶的组织形态发生。因此，在许多情况下他们的研究有直接影响，为了解人类疾病（见表1）1-5 。规模在亚细胞，这些细胞形态的变化，产品范围从基因表达的信号转导，细胞极性，细胞骨架重塑和膜贩运活动。因此， 果蝇胚胎不仅提供了背景下，以评估细胞形状的变化，因为这涉及到组织的形态发生，但也提供了一个完全的生理环境研究的亚细胞形状的细胞的活动，。 这里描述的是该协议设计的图像称为cellularization一个特定的细胞形态变化。 Cellularization是一个戏剧性的质膜增长过程，并最终转换成合胞胚胎细胞胚。也就是说，在有丝分裂周期14相间，质膜同时invaginates〜6000 cortically挂靠核周围产生原发性上皮细胞表。 cellularization违反上述建议，是不是驱动由肌球蛋白收缩6，但燃料，而不是主要从7个内部存储的膜胞吐。因此，cellularization是一个优秀的系统研究在细胞形态的变化，需要质膜内陷或扩建，如细胞分裂或横管（T型肾小管）在肌肉形态发生，膜贩运。 请注意，这个协议是很容易应用到其他细胞形状的变化，在果蝇胚胎成像，并且只需要轻微的适应化修改，如改变胚胎收集的阶段，或使用"胚胎胶"，安装在一个特定的方向（表胚胎1）8-19。在所有情况下，工作流程基本上是相同的的（图1）。克隆和转基因果蝇的标准方法用于制备稳定的飞股票，表达利益的一种蛋白，融合，绿色荧光蛋白（GFP）或它的变种，和这些苍蝇提供了一种可再生的胚胎来源。另外，荧光蛋白/探针直接引入飞胚胎，通过简单的显微注射技术 9-10 。然后，根据发展的事件和细胞形状的变化进行成像，胚胎收集和形态解剖显微镜上演，并最终定位和安装时间推移共聚焦显微镜成像。

Watch this Scientific Journal Video about Imaging Cell Shape Change in Living Drosophila Embryos at JoVE.com

Imaging Cell Shape Change in Living Drosophila Embryos

果蝇，果蝇，早期发展的特点是由许多细胞形状的变化，以及成像方法适合。本文将描述活果蝇胚胎的共焦成像所需的基本工具和方法，将集中在细胞形态变化称为cellularization。

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

imaging-cell-shape-change-in-living-drosophila-embryos

Research

JoVE Journal

Biology

Imaging Cell Shape Change in Living Drosophila Embryos

14.2K 次观看.  Baylor College of Medicine (BCM). 果蝇，果蝇，早期发展的特点是由许多细胞形状的变化，以及成像方法适合。本文将描述活果蝇胚胎的共焦成像所需的基本工具和方法，将集中在细胞形态变化称为cellularization。

视频: Imaging Cell Shape Change in Living Drosophila Embryos

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

从Midgastrula舞台果蝇胚胎神经文化的制备

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

科研

生物学

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

编制果蝇实时成像的胚胎使用悬滴协议

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

果蝇胚胎的体内离心

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

直立成像果蝇胚胎

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

原代细胞培养从果蝇原肠胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

kinesin的分离和纯化果蝇胚胎

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

龙虎斗动粒蛋白的动态行为与染色体运动，在生活学习有丝分裂关卡<em&gt;果蝇</em&gt;合胞胚胎

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

影像生活细胞的形状变化果蝇胚胎

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