我们感谢提供材料测试Sciara coprophila Ilyanassa obsoleta体内离心，分别苏珊Gerbi，海蒂史密斯和大卫兰伯特。我们感谢的技术和手稿的意见Welte实验室的成员。 在体内的离心检测的发展和细化支持NIGMS授予GM64687鱼鳔。图像如图所示​​。 1和图。 5E，F察看2，经许可后转载。

1）准备琼脂平板 概述：此协议采用琼脂平板上，两个不同的用途。首先，琼脂平板作为鸡蛋征收面;苹果汁琼脂刺激产蛋。其次，在琼脂嵌入式胚胎举行，在一个固定的方向时板离心;一个足够高的浓度，琼脂是必要的，以防止在离心瓦解板。为了简便起见，都使用单一类型的苹果汁琼脂聘用。 材料<ul><li>琼脂（25克） </li><li>蔗糖（25克） </li><li>苹果汁（250毫升） </li><li>尼泊金中号原液（5克甲基- P -羟基苯甲酸在50 mL无水乙醇），作为防腐剂</li><li>培养皿：离心（60x15mm）和鸡蛋收集（60x15毫米或100x15毫米，根据在第2步使用的飞笼） </li></ul>设备<ul><li>热板</li><li>两个锥形瓶</li></ul><ol><li>在锥形瓶中，结合25克琼脂和750毫升DH 2 O。高压灭菌液体周期为50分钟。与此同时，准备苹果汁溶液（步骤1.2）。 </li><li>在另一锥形瓶中，结合25克蔗糖和250毫升的苹果汁。热板的热（约55℃）溶解蔗糖。然后添加15毫升尼泊金中号溶液。避免较高的温度，因为它们会导致尼泊金打破。 </li><li>一旦琼脂溶液冷却烧瓶中可以通过手工处理，将二者结合起来的解决方案，调匀。热板搅拌，直到混合均匀。 </li><li>倒入培养皿（约0.5厘米高）的解决方案。 1公升的解决方案就足够了大约100个​​小型（直径60毫米）或40大直径（100毫米）菜肴。 </li><li>琼脂平板应干几个小时或在室温下过夜，否则会太湿的使用。对于长期贮存，保持他们包裹在塑料箱或培养皿袖子在4 ° C。 </li></ol> 2）收获胚胎 概述：在发展的第3小时（室温）， 果蝇胚胎的单个细胞（不计的种系的前体，向后位于杆细胞）。当这些阶段的正确导向胚胎离心，密度单独的主要细胞器沿长轴鸡蛋。 cellularization阶段，这个大的单细胞转换成数以千计的小细胞，离心势力不会破裂，这些细胞。主要细胞器的成功分离，因此，它是关键离心机尚未完成cellularization的胚胎。 材料<ul><li>苹果汁琼脂平板</li><li>酵母膏（花生酱的一致性，与水混合干酵母） </li></ul>设备<ul><li>蝇笼：市售（见下文）或自制的1 </li></ul><ol><li>成人苍蝇都保存在苹果汁琼脂平板贴在底部的笼子。使用培养皿中使用的笼子适当的大小。 </li><li>要启动一个鸡蛋收集，准备一个新鲜的苹果汁琼脂平板，并涵盖了部分与酵母膏。果蝇吃酵母，尤其是酵母提供营养蛋的生产。酵母和苹果汁琼脂的存在也引起产蛋。 </li><li>交换旧与新的琼脂平板，飞笼倒在板凳上的顶部撞了几次。苍蝇将下降到谷底，并简要地迷失方向。这就给了你几秒钟，迅速取出一个琼脂平板，并更换新鲜。这种交流需要一些练习，和细节上笼的类型取决于。 </li><li>从以前的交配内部邮袋（精囊）发布的精子使卵受精的雌性果蝇储存精子。当美联储和不受干扰，雌虫受精后不久，蛋，产蛋的时间，从而标志着受精和发展开始的时间。当条件不理想，女性持有铺设前的受精卵变量倍。为了尽量减少这样的MIS上演胚胎的数量，最好是放弃第一个工作日的集合（“预收款”的30至60分钟就足够了）。鲜酵母的存在导致女性打下这些举行的蛋，和随后的蛋集合往往是由鸡蛋为主沉积在受精后不久。 </li><li>鸡蛋收集长达3个小时，这取决于所需的萌芽阶段。由于是在室温下cellularization〜3.5小时后完成，收集时间较长用处都没有。最一致的分层实现了幼胚，常规实验，我们赞成征收时间较短（1小时或更少）。 </li><li>有时苍蝇卡住酵母或琼脂平板表面。用镊子取出。 </li></ol> 3）从胚胎取出蛋壳 概述：外层（绒毛膜）（卵黄膜）为主制成的蛋白质和内层蜡： 果蝇胚胎是由两个保护层覆盖。他们免受机械侮辱和干燥的胚胎。绒毛膜有两个扩展（鸡蛋丝或背附属物）作为独特的结构，很容易可见。在这一步，我们将删除50％的漂白粉浸泡鸡蛋的蛋壳。胚胎绒毛膜一旦被删除，变得透明，在透射光下，允许选择具体的萌芽阶段，离心。由于绒毛膜也会干扰许多离心后的程序（例如，固定在第6步），去除绒毛膜现在将允许快速处理后。 50％的漂白治疗绒毛膜会溶解在几分钟之内，但它不会伤害胚胎。卵黄膜保持漂白了。我们将继续漂白处理，直到鸡蛋丝不再可见。之后，我们将单独浇通过一个很小的筛子（丝网制成的一篮子）的胚胎/漂白浆漂白的胚胎。至关重要的是不要急于漂白的曝光步骤。如果漂白洗掉过早，以后的步骤（例如，去除卵黄膜下面固定）可能会受到损害。 材料： <ul><li> 50％漂白水喷瓶（一个卷一个卷商业漂白DH 2 O ） </li><li>水枪瓶与DH 2 O </li><li>纸巾</li></ul>设备： <ul><li>国产钢丝篮，不锈钢丝网张。为了使篮，切〜2 × 2厘米的平板平方和缩进在他们中间。 </li><li>用镊子将举行丝篮</li><li>解剖显微镜设置为透</li></ul><ol><li>琼脂平板上，并放置在解剖显微镜下观察胚胎透。确保识别鸡蛋丝（见图2A）。 </li><li>水枪50％的琼脂平板上，漂白剂，覆盖的胚胎。偶尔搅动琼脂平板胚胎周围的漩涡中的漂白。 </li><li>一旦鸡蛋丝不再可见（通常在3-5分钟，但时间可能有所不同），绒毛膜已成功删除。 </li><li>在接下来的步骤，丝网篮将被用来作为筛分开漂白剂的胚胎。用镊子抓住篮筐，缓缴琼脂平板倒盖。封面将作为水库捕获的漂白剂滴通过丝网。 </li><li>通过琼脂平板丝网倒入浆的漂白/胚胎。 </li><li>如果大量的胚胎留在琼脂平板上，喷出的dh 2到盘子和倒通过丝网的dh 2 /胚胎浆。如果大量的胚胎溅入水库，通过丝网倒入水库的内容。 </li><li>丝网底部的触摸纸巾吸干了任何多余的液体。然后喷射到丝网DH 2 O冲洗掉剩余的漂白。如上所述，盖板可以被用来作为水库恢复不小心洗掉任何胚胎。您可以最大限度地减少损失，指向沿着篮筐周围喷瓶水流的胚胎。常见吸干多余的液体。 </li><li>重复此清洗步骤五到十倍，已被删除，直到所有的漂白剂。如果篮筐仍然气味的漂白剂，洗衣机应该继续下去。 </li></ol> 4）在琼脂安装胚胎 概述： 果蝇卵是远远长于宽（〜180微米）（〜500微米）。为了获得最大的和一致的过程中离心分离细胞器，我们东方的胚胎，其前结束对旋转中心的点（图1，3）。通过这种方式，最轻的细胞内的结构脂滴积聚于前年底在所有的胚胎，而非常致密的结构（蛋黄组件）附近的后结束积累。胚胎苹果汁琼脂小垂直孔插入，坚持前结束。琼脂保持在一个固定在离心方向的胚胎。 设备： <ul><li>持针器</li><li>钨针，针安装在持有人从典型的方向向后，使钝端伸出操纵胚胎</li><li> P200的移液器</li><li>解剖显微镜设置为transilluminatioñ </li><li>飞刷（小油漆刷用来排序成年果蝇） </li></ul>材料： <ul><li>海卫盐溶液（TSS）：4G氯化钠，0.3毫升TRITON X - 100，填写至1000毫升（可作为一个10倍的股票解决方案与DDH 2 O ） </li></ul><ol><li>洗篮筐的胚胎与TSS和到一个空的培养皿。胚胎沉底。解剖范围转移到TSS中含有胚胎的培养皿，观察和透（胚胎将类似于如图4所示）。 </li><li>使用P200的移液器，选择所需阶段的胚胎，将它们转移到一个小琼脂平板。特定阶段可以很容易地被认可的视觉（图4;如需详细资讯，请参阅1） 。因为胚胎的老化和长期淹没在TSS（长于30分钟）可能会影响发展迅速。 </li><li>取出用移液器和Kimwipe最TSS的。琼脂表面应稍湿润，这使得它更容易推胚胎。但是，不应该其余板的任何地方，在离心过程中多余的液体，因为会导致胚胎幻灯片孔液体池。 </li><li>用针，垂直孔捅到附近的地方位于一个胚胎的琼脂。是完全垂直的万佛洞，这是很难，因为你必须保持在一个角度的针能够同时在显微镜下观看。一个轻微的倾斜针实际上是有利的，因为这将创建洞的一侧有轻微的抑郁症/树丛，使胚胎很容易滑动和成孔。这是足够的琼脂穿刺琼脂表面，因为是软的，足够的，它可以在不损害进一步推到一个孔中插入一个胚胎一次是部分。 </li><li>确保你认识的胚胎的前部和后部的结束，胚胎应推入孔方向一致，与前年底通常可达。前结束的卵黄膜是由专门的结构特点，珠孔（图2），通过它在受精过程中精子进入。 </li><li>使用的针头钝端，你可以把对胚胎沿着琼脂和东方/机动走向刺破孔后结束。对孔，然后反推前结束。这应该很容易滑动沿在穿刺产生的轻微的抑郁症孔进入胚胎。由于胚胎倾斜，推动更深的成孔。 </li><li>全面推开时，胚胎通常已经相当垂直。如果没有，你可以更垂直对齐插入的胚胎由侧向推它。如果孔是不是太大，胚胎的位置将稳定整个离心琼脂举行。 </li><li>通过实践，它是可以嵌入到每板100-250胚胎。这将取决于特定的应用程序需要多少：如果胚胎用于实时成像或捐助者移植实验中，只有少数需要。如果胚胎加以固定和免疫染色，一小部分将失去在离心后处理，并开始与更高的数字。请记住，胚胎继续发展过​​程中安装这样一个狭窄的发展阶段，如果需要，安装需要的总时间要尽量短。 </li><li>如果有任何剩余的胚胎，不嵌入，用蘸飞刷从板块中删除。沾飞刷成液体（例如1xTSS），仔细地擦拭板的顶部（下解剖范围看）扫描任何剩余的胚胎，而胚胎的刷子清洗浸泡成液体的刷一遍。 </li></ol> 5）离心和胚胎复苏 概述：板转移到摆动水桶一个Sorvall RT7加离心机离心30分钟在4000转，这相当于约3000克。离心后，板与TSS的覆盖。用针，胚胎被挖了出来在琼脂上的孔，并通过移液器转移到适当的船只。 设备： <ul><li>针持针器（如前） </li><li> Sorvall RT7加RTH750转子和摆动水桶，或相当于2 </li><li>解剖显微镜设置为透</li><li> P200的移液器</li></ul>材料： <ul><li> TSS </li></ul><ol><li>传输板，离心机，琼脂的一面向下摆动水桶。确保离心机的均衡。 </li><li>离心设置：温度= 4-10 ° C，转速= 4000时间= 30分钟。 </li><li>经过离心，琼脂平板解剖显微镜下再次和覆盖层的TSS板。 </li><li>使用B针水汽年底，掏的胚胎，其孔。他们将在TSS的浮动，但仍将淹没。 </li><li>胚胎会出现明显分层，棕色第一个在年底前，一个明确的中部地区，一片漆黑，在后部的灰色地带（图2，5A）。丢弃的胚胎，未能表现出鲜明的分层的。 </li><li>使用P200的移液器，TSS到一个新的船只的层次胚胎转移，如闪烁瓶或离心管，根据不同的应用。 </li></ol> 6）进一步处理根据不同的应用，胚胎也将随之被视为以不同的方式。 <ol><li>明场或epifluorescence显微镜直接观察，胚胎在缓冲区转移到玻片上，并安装在一个22x22毫米＃1.5盖玻片＃1盖玻片18 × 18毫米间隔。对于长期观察，它可能是必要的，以取代卤烃油27缓冲区。 </li><li>如果是固定的，胚胎，他们应该在步骤5.6转移到闪烁瓶中。然后，他们可以是固定的，使用标准的固定技术 1 。这些技术通常采用在庚烷甲醇乳化搅拌，去除卵黄膜。注意离心胚胎这一devitellinization步骤的效率是低的。因此，建议开始与实际许多胚胎或手动删除卵黄膜1。 </li><li>如果胚胎将被使用（例如，删除一个特定的细胞器层）的离心胚胎移植实验，他们转移到平 ​​板装上盖玻片，使用标准程序3 uncentrifuged胚胎。离心诱导分层是稳定的几十分钟。 </li></ol> 7）代表性的成果如果离心工作不如预期，主要胚胎细胞器将理清密度2。例如，低密度脂滴积聚于前年底，高密度的蛋黄囊泡会积聚在后结束（图1，2）。脂滴和蛋黄囊泡存储脂类和蛋白质的细胞器。 透密度依赖分层的确认是通过解剖或复式显微镜下，通过目视检查活胚胎。胚胎应该出现在图5A：脂滴将在很前的提示（“脂盖”），形成一个坚实的的棕色层;卵黄积累后的最终结果将在一个黑暗的灰色地带。代表其他细胞器和细胞质了广阔的明确区分开这两个暗层。如果胚胎后cellularization离心，分层将微乎其微（图5C）。 如果epifluorescence显微镜，这些生活，离心胚胎紫外线激发下可以检查。在这些情况下，蛋黄，显示了强烈的蓝色自体荧光（图5B）。一个在后极autofluorescent材料的致密层，表示成功离心后结束的标记。 请注意，这些层的外观将发生急剧变化，如果胚胎是固定的。例如，当胚胎是由固定的标准热或甲醛固定庚烷甲醇devitellinization 1，成为半透明的脂滴和脂质层出现发白和蓬松的，由明亮的光镜，而不是黑褐色（图5D ）。卵黄自体荧光是由固定部分或完全被毁，成为少得多激烈，甚至检测不到。 如果体内离心突出一个特定的细胞器，它是有帮助的标签，细胞器与荧光标记进行确认。例如，YFP的融合蛋白定位到线粒体，ER或高尔基被描述4。在离心胚胎，这些标志物堆积在沿前后轴（图1）的特征位置。最后，某些组蛋白的融合蛋白本地化脂滴和原子核 2，因而可以用来标记这些细胞车厢（图5，E，F）以及监控胚胎阶段（标记细胞核的数量） 。可从布卢明顿果蝇股票中心（http://flystocks.bio.indiana.edu/）飞株表达在本段中提到的标记。对于组蛋白H2Av，GFP和MRFP变种是5， 6 <img src="/files/ftp_upload/2005/2005fig1.jpg" alt="图1" /> 图1通过离心分离细胞器（修改后 2） 。离心型胚胎在不同的分层结果（耀眼的光芒）。主要细胞器的积累特征的立场，沿前（上） - 后（下）轴：脂滴（固定胚胎尼罗红染色检测）;内质网，高尔基体，线粒体（活胚胎通过在YFP的标记检测主要文本）;蛋黄（自体荧光检测）。共聚焦显微镜，荧光图像被收购。 <img src="/files/ftp_upload/2005/2005fig2.jpg" alt="图2" /> 图2原理图描绘的胚胎。 答：鸡蛋与鸡蛋壳和突出的鸡蛋丝（背附肢。）由于鸡蛋壳是不透光的，出现鸡蛋均匀黑暗。 B.与蛋壳的蛋删除。前端（左），珠孔是可见的。根据发展阶段，胚胎内的卵黄膜会出现均匀暗棕色或显示不同的结构。参见图4-3的例子。 C.离心胚胎，在这里描述的协议为导向。脂滴的积累作为一个棕色的“帽子”，在珠孔端;蛋黄形成了一层灰色或附近的另一端。 <img src="/files/ftp_upload/2005/2005fig3.jpg" alt="图3" /> 图3。示意图描述的过程。左：没有蛋壳的胚胎放在琼脂平板上，用针推入孔在琼脂。这种安装在所有胚胎的一致方向的结果，与前结束。右：离心后，胚胎是分层的。琼脂覆盖TSS（蓝色），胚胎从琼脂针收回。 TSS的暂停后，胚胎可以拿起一个移液器。 <img src="/files/ftp_upload/2005/2005fig4.jpg" alt="图4" /> 图4。亮光影像生活果蝇胚胎，他们怎么会出现在步骤4.1相似。胚胎标准方向：前年底到左边，正确的，背的一面朝上，腹侧下来后结束。一个早期胚胎发育的时间间隔短片，作为本议定书所附的视频的一部分。 A.卵裂阶段的胚胎出现均匀的棕色。像这样的阶段是理想的体内离心。 B.胚胚胎有一个清晰的轮辋的出现在各方面相似。这个清晰的边缘，部分原因是由于积累的原子核在细胞膜和部分由于卵黄泡和脂滴7外来运输。这个明确的外围扩展，直至接近 cellularization 1月底。胚胎仍然可以成功地通过离心分层cellularization 8月底之前，虽然蛋黄和原子核的层次感不卵裂阶段一致。 C.后cellularization，胚胎出现不对称;成为独特的背，腹两侧，以及各种褶皱的发展。显示胚胎在早期生殖波段扩展。在这些阶段，离心分层的主要细胞器不再有效。 <img src="/files/ftp_upload/2005/2005fig5.jpg" alt="图5" /> 图5。离心胚胎。在此图中的所有胚胎离心其前结束，并在该方向显示。 A.亮光的形象，一个成功的离心胚胎。在很前结束的棕色层是由于脂滴的积累。一个广阔的后部附近的灰层是由于蛋黄囊泡。经常有一个来历不明的，下面的蛋黄层明确区。淡黄色的区域往往是明显的蛋黄层之上，它可能是线粒体。可溶性蛋白的发现整个清晰的层之间的脂滴和蛋黄2。 B.胚胎在一个由紫外线激发下epifluorescence显微镜观看。蛋黄的蓝色自体荧光的特性，有助于确定后结束。 C.胚胎生殖波段扩展，即在离心后cellularization。蛋黄仍可能积聚在后结束，但另有分层是最小的。 D.成功分层胚胎后，热固定。脂滴层出现白色，蓬松，而不是在不固定的胚胎暗棕色（一）的。 E.＆F胚胎表达His2Av - GFP和成像共聚焦显微镜（修改后2）。电子邮件：离心裂解阶段; His2Av - GFP似乎只标记的脂滴层，因为这个时候形成的几个核。根据焦平面上，细胞核可刚下液滴层可见（此处未显示）。传真：离心胚阶段; His2Av - GFP标记的脂滴层（顶部）和细胞核（在液滴层以下的广阔地带）。

<ol>
	<li>Wieschaus, E., N&#252;sslein-Volhard, C., Roberts, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+at+embryos.">Looking at embryos.</a> Drosophila: A Practical Approach. , 179-214 (1998).</li><li>Cermelli, S., Guo, Y., Gross, S. P., Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lipid+droplet+proteome+reveals+that+droplets+are+a+protein+storage+depot.">The lipid droplet proteome reveals that droplets are a protein storage depot.</a> Curr Biol. 16, 1783-1795 (2006).</li><li>Kiehart, D. P., Crawford, J. M., Montague, R. A., Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+microinjection+of+Drosophila+embryos.">Quantitative microinjection of Drosophila embryos.</a> Drosophila Protocols. , 345-359 (2000).</li><li>LaJeunesse, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+new+Drosophila+markers+of+intracellular+membranes.">Three new Drosophila markers of intracellular membranes.</a> Biotechniques. 36 (784-788), 790-790 (2004).</li><li>Schuh, M., Lehner, C. F., Heidmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+Drosophila+CID/CENP-A+and+CENP-C+into+centromeres+during+early+embryonic+anaphase.">Incorporation of Drosophila CID/CENP-A and CENP-C into centromeres during early embryonic anaphase.</a> Curr Biol. 17, 237-243 (2007).</li><li>Clarkson, M., Saint, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+His2AvDGFP+fusion+gene+complements+a+lethal+His2AvD+mutant+allele+and+provides+an+in+vivo+marker+for+Drosophila+chromosome+behavior.">A His2AvDGFP fusion gene complements a lethal His2AvD mutant allele and provides an in vivo marker for Drosophila chromosome behavior.</a> DNA Cell Biol. 18, 457-462 (1999).</li><li>Welte, M. A., Gross, S. P., Postner, M., Block, S. M., Wieschaus, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+regulation+of+vesicle+transport+in+Drosophila+embryos:+forces+and+kinetics.">Developmental regulation of vesicle transport in Drosophila embryos: forces and kinetics.</a> Cell. 92, 547-557 (1998).</li><li>Tran, S. L., Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=unpublished+observations.">unpublished observations.</a> , .</li><li>Rothwell, W. F., Sullivan, W., Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+analysis+of+Drosophila+embryos.">Fluorescent analysis of Drosophila embryos.</a> Drosophila Protocols. , 141-157 (2000).</li><li>Guo, Y., Jangi, S., Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle-specific+Control+of+Intracellular+Transport:+Distinctly+Targeted+Isoforms+of+the+Regulator+Klar.">Organelle-specific Control of Intracellular Transport: Distinctly Targeted Isoforms of the Regulator Klar.</a> Mol Biol Cell. 16, 1406-1416 (2005).</li><li>Meyer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overlapping+Functions+of+Argonaute+Proteins+in+Patterning+and+Morphogenesis+of+Drosophila+Embryos.">Overlapping Functions of Argonaute Proteins in Patterning and Morphogenesis of Drosophila Embryos.</a> PLoS Genet. 2, 1224-1239 (2006).</li><li>Shubeita, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+motor+copy+number+on+the+intracellular+transport+of+kinesin-1-driven+lipid+droplets.">Consequences of motor copy number on the intracellular transport of kinesin-1-driven lipid droplets.</a> Cell. 135, 1098-1107 (2008).</li><li>Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+lipid-droplet+transport+by+the+Perilipin+homologue+LSD2.">Regulation of lipid-droplet transport by the Perilipin homologue LSD2.</a> Curr. Biol. 15, 1266-1275 (2005).</li><li>Phadnis, N., Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unpublished+Observations.">Unpublished Observations.</a> , .</li><li>Phadnis, N., Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unpublished+Observations.">Unpublished Observations.</a> , .</li><li>Morgan, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+XXII:+The+redistribution+of+the+visible+materials+of+the+egg+by+centrifuging.">Chapter XXII: The redistribution of the visible materials of the egg by centrifuging.</a> Experimental Embryology. , (1927).</li><li>Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unpublished+Observations.">Unpublished Observations.</a> , .</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=Abnormal+oogenesis+and+embryogenesis+resulting+from+centrifuging+Drosophila+melanogaster+females.">Abnormal oogenesis and embryogenesis resulting from centrifuging Drosophila melanogaster females.</a> J Embryol Exp Morphol. 40, 65-81 (1977).</li></ol>

关键步骤 确保琼脂浓度足够高 ，如果它太低，琼脂将瓦解在离心过程中，胚胎会爆裂或随机取向。如果琼脂的浓度只是稍微过低或琼脂是湿的（不够晒干或步骤4.3中清除多余的TSS的不足），胚胎会浮在离心其孔，成为不正确导向。琼脂倒入如果过于分散，它也将崩溃或裂纹在离心。 确保胚胎不能超越cellularization阶段的先进，否则，细胞器的分离将无法工作（图5C），因为细胞器将成为成千上万的单个细胞内固定。在年轻的一个细胞阶段的胚胎，细胞器是自由迁移大的距离，根据他们的特点密度和积累。为了避免分析胚胎太旧，确保（i）包括一个预收款，让女性躺在老蛋（步骤2.4），（二）收集3小时或更少的胚胎（步骤2.5），（三）直观地选择前cellularization阶段的胚胎嵌入到琼脂（步骤4.2），（四）保持嵌入时间短，以防止通过发展cellularization（步骤4.6）结束，（五）正确丢弃的胚胎，没有层（步骤5.6） 。 不要吝啬离心时间 ，虽然有些胚胎将显示很好的分层，即使只有10分钟的离心后，分离是从胚胎到胚胎的不一致。 30分钟旋转给予了高度一致的结果。此外，离心时间较长，不再层是稳定的离心后已经结束。这一点很重要，这些应用程序，它需要时间处理，使用前胚胎进一步（例如，如果他们需要处理的移植实验，或安装共聚焦分析）。 可能的修改 蛋收集和绒毛膜去除许多不同的协议中存在的步骤2 和3 1，3，9，和会的工作，以及这里的描述，只要使用的胚胎是年轻，鸡蛋产量高，分数MIS上演的胚胎是最小化。 该协议前后对齐定位所有胚胎后到底是埋在琼脂和前结束（图3）。这个方向是一致性好，因此，它有可能使用年底指出在离心5）（图1，作为一个具有里程碑意义的珠孔。然而，实践中，它很容易挑选出的脂滴和蛋黄层亮视野显微镜的独特的外观离心胚胎的方向。然后安装任何一端的胚胎成为可能，这种灵活性加快了安装过程。 无向离心的协议中最耗费时间和技术上具有挑战性的方面是安装在琼脂胚胎（步骤4 ）。它需要触及每个胚胎两次（一次插入孔内，并一次来恢复它，离心后）。作为替代方案，可以在步骤4.1传输与TSS的离心管中充满的胚胎。当这些管子中的微量离心（13,000 RPM，10-30分钟），胚胎通常也会层以及10-13。这种变化可以处理数百个胚胎，在同一时间，速度非常快。然而，层叠不作为一致;一小部分的胚胎不发展，尽管不同的层次要高得多的离心势力（〜16,000 G），比上述协议中的应用。更具有挑战性的是，胚胎的方向是可变的，人们可以观察到不同的角度主要胚胎轴分离。这种可变性，需要实验者进行排序后，在一个特定的胚胎是如何安排在离心管中离心。蛋黄自体荧光标记胚胎的最密集的部分使用，这往往是可能的，但它需要大量的实践和更多的判断做可靠。如果有足够的胚胎样品中，一种可能是能够理清这些实例在胚胎发生像图安排。 1。 离心他们的蛋壳中的胚胎很可能在步骤4琼脂中嵌入胚胎不删除绒毛膜第2。在这种方法中，胚胎收集盘上都覆盖着卤烃油，而不是50％漂白步骤3.2（卤烃变成绒毛膜半透明）。选择适当阶段的胚胎，并转移到一个新的琼脂平板上，用小镊子抓住镊子，损害的只有鸡蛋丝胚胎适当是可以避免的。胚胎是嵌入在步骤4.4至4.8％，与前鸡蛋丝琼脂孔伸出。蛋壳被删除50％漂白处理后的胚胎已经离心，在步骤5.4的琼脂挖。在鸡蛋壳的存在离心可提高固定后devitellinization率（步骤6.2），但选择正确的离心阶段（步骤4.2）是更具挑战性的。 应用胚胎离心共定位实验，即进行测试，如果一种特定的蛋白质定位于一定的主要细胞器是非常有用的。例如，Klar蛋白是目前对早期胚胎的脂滴，但这种本地化是具有挑战性的，以展示完整的胚胎。在某种程度上，这是因为这两个Klar puncta和脂滴丰富，使得虚假的共定位很难排除在胚胎外围10。然而，离心浓缩成一个独特的层脂滴，因此，共定位Klar信号变得明显10。 类似的分析，明确地表明了对其他蛋白质的本地化脂滴8日，13日至 15日，包括令人惊讶的例子，如某些组蛋白2的一种蛋白质是目前普遍存在的情况下，但丰富的脂滴8。作为胚胎发育阶段（步骤4.2），可以选择在视觉上，它甚至可以检测发育调控的蛋白质招聘脂滴8，15。测试与脂滴共定位是特别容易，因为脂滴堆积在视觉上不同的“帽子”，所以没有进一步的标记或染色，以确定这一层。事实上，我们的实验室，因为它的易用性和潜在引人注目的视觉效果（图1，图5 D，E），采用这种技术，现在通常作为第一个测试候选人蛋白是否是本地化的脂滴。原则上，类似的共定位研究工作图的其他细胞器。 1，并有可能为他人（包括细胞内寄生虫），其分布在离心胚胎还没有被记录在案。 由于离心浓缩成严密乐队的细胞器，它有利于在这些细胞器丰富的一小部分隔离。这种方法已经被用来恢复到捐助者的胚胎移植2的脂滴。它也是可能的生化分析丰富的一小部分（例如，通过切断胚层固定后15）。 描述的方法，这里已经超越果蝇胚胎的应用，如可用于离心分层许多不同的物种（包括青蛙，线虫，被囊动物，环节动物，海胆，软体动物，腔肠动物）16鸡蛋。在我们自己的实验室，我们使用以上家蝇家蝇胚胎协议，并已聘用在胚胎从真菌GNAT Sciara coprophila 17日和蜗牛Ilyanassa obsoleta 17分析的无向离心。最后，这种方法并不局限于胚胎：离心等大型的细胞，如卵母细胞（例如， 在 果蝇2，18日和马利筋错误Oncopeltus fasciatus 17），也可以分层。对于不同的试样，确切的离心条件下需要进行优化：例如，在3000克10分钟是足够的，有效地进行 分层胚胎家蝇2，如果离心更长的时间，胚胎往往打破固定和免疫期间除了。

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>60x15 mm Petri dishes</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>#35-1007</td>
<td>From Falcon</td>
</tr>
<tr>
<td>Wire mesh</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>CX-0150</td>
<td>stainless steel type 304; mesh size #150 x150, wire diameter: 0.0026 inches</td>
</tr>
<tr>
<td>Tungsten needles</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>10130-10</td>
<td>0.25 mm</td>
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<tr>
<td>Moria Nickel Plated Pin/Needle Holder</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26016-12</td>
<td>&nbsp;</td>
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<td>Fly Cages</td>
<td>&nbsp;</td>
<td>Genesee Scientific</td>
<td>59-100 or 59-101</td>
<td>For 65 mm or 100 mm diameter Petri dishes, respectively</td>
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<td>Halocarbon Oil 27</td>
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in-vivo centrifugation of drosophila embryos

净化和隔离不同类型的细胞器的一个重大战略，是他们彼此分开的基础上浮力密度的差异。然而，当细胞被破坏之前，在这个非母语的环境中的离心，蛋白质和细胞器往往不适当地坚持对方。在这里，我们描述一个单独的细胞器的方法，完好，生活果蝇胚胎中的密度。收获人口笼cellularization前的早期胚胎，并删除其外层的蛋壳是用50％漂白粉治疗。胚胎，然后转移到一个小的琼脂平板和插入，结束后第一，在琼脂小垂直孔。包含嵌入式胚胎的板是离心30分钟3000克。琼脂支持胚胎，并保持在一个确定的方向。之后，胚胎挖的琼脂用钝针。 离心分离成不同的层次的重要细胞器，一个分层容易被亮视野显微镜可见。一些荧光标记确认活胚胎的成功分层。与某些细胞器相关的蛋白质会在一个特定的层丰富，展示共区域化。生化分析或移植到捐赠卵子可以恢复单个图层。这种技术适用于在其他大的细胞，包括品种多样的卵和卵母细胞的细胞器分离。

Watch this Scientific Journal Video about In-vivo Centrifugation of Drosophila Embryos at JoVE.com

In-vivo Centrifugation of Drosophila Embryos

我们描述了独立的细胞器密度的方法，在生活<em&gt;果蝇</em&gt;胚胎。胚胎中嵌入琼脂，离心。这种技术产生沿前后胚胎轴重复性的主要细胞器的分离。这种方法有利于共定位实验和产量的细胞器组分生化分析和移植实验。

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 ...

in-vivo-centrifugation-of-drosophila-embryos

Research

JoVE Journal

Biology

In-vivo Centrifugation of Drosophila Embryos

18.3K 次观看.  University of Rochester. 我们描述了独立的细胞器密度的方法，在生活果蝇胚胎。胚胎中嵌入琼脂，离心。这种技术产生沿前后胚胎轴重复性的主要细胞器的分离。这种方法有利于共定位实验和产量的细胞器组分生化分析和移植实验。

视频: In-vivo Centrifugation of 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

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 ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

为介观荧光体层摄影术在体内成像的发展果蝇</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

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 ...

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 ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

RNAi的干扰双链RNA注射到果蝇胚胎

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called 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 ...

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 ...