我们感谢他们的贡献，这些协议的发展Nicki福克斯和阿洛伊西亚施密德。这项工作是支持由NIH RO1授予KZ​​，NS28182。

简介 果蝇胚胎胚胎发育的阶段，14和17之间，可以很容易地解剖产生"鱼片"的准备工作。在这些准备工作，中枢神经系统（CNS）运行中间，是由身体墙壁两侧。肠道中被删除。当抗体染色，鱼片允许更好的可视化的中枢神经系统和体壁结构（如电机轴突，肌肉，周边感觉神经元（PNS）的，气管）比整个安装的胚胎，因为没有组织之间的准备和干预盖玻片，因为鱼片是平的，允许横跨体壁结构，在一个单一的焦平面的可视化。许多不同表型已使用这种制剂的研究。在大多数情况下，鱼片是由固定，抗体染色的整体安装胚胎清扫生成。这些固定的筹备工作正在产生以下步骤:1）绒毛膜去除漂白剂;结算甘油5）; 3）卵黄膜用甲醇去除; 4）采用免疫组织化学或免疫抗体染色2）与多聚甲醛/庚烷固定6）清扫与钨针。染色这些"固定解剖"的详细协议提供参考。 [1]。 固定的解剖，但有一些缺点。首先，它往往是难以理清固定，染色突变体（GFP负）的股票或突变，其中超过GFP的平衡器平衡的杂交胚胎，即使抗GFP用于检测。这是由于多种因素，包括产妇的GFP表达。例如，我们发现它几乎是不可能的排序从平衡的第三号染色体的股票，无论是肌动蛋白GFP或犰狳（ARM）- GFP的平衡器固定，染色的纯合子突变胚胎的。其次，它是非常耗时的生成高质量的固定解剖。每小时10-15是一样快，因为大多数人能做到这一点。第三，一些抗体不染色以及在固定的解剖，要么是因为抗体的抗原决定簇是敏感的修复，或因为一种抗体，污渍的内部和外部的结构是"浸泡"的外部结构，不渗透到内部结构（ 如对fasciclin三（Fas3））的抗体。第四，现场染色检测配体的表达与受体融合蛋白不能做固定的筹备工作。 自2002年以来，本集团一直在进行缺乏症（DF）和异位表达屏幕识别RPTP的配体。为了做到这一点，我们开发的精简协议活胚胎剥离和染色包含数百个平衡线路的集合。孤儿受体的配体与受体融合蛋白的染色是一个专门的应用程序，是不是由许多团体。然而，许多团体使用抗体染色鱼片胚胎表型的可视化。通过我们的这些方法的发展，我们已经得出结论，这是相当多的效率比固定清扫现场清扫研究股票的大集合的表型。我们用现场清扫到600多个DFS运动神经，中枢神经系统和肌肉的表型特征，也具有神经系统，超过400种不同的细胞表面的异位表达和分泌蛋白（胡等人在准备的产生表型。香港[ 等 。准备）。 经过多年的发展我们已经使用了现场解剖协议。的作者之一（KZ）首次推出生活NIPAM帕特尔，谁当时在科里 - 古德曼的实验室研究生20余年前的夹层。在最近几年中，我们使用这种方法，染色与反Fas3 [2]，但雇用的所有其他实验固定解剖的中枢神经系统。在1999年，在本集团的博士后，阿洛伊西亚施密德，然后介绍我们的生活与夹层玻璃针，她用双链核糖核酸，注射胚胎[3，4]研究表型。当我们开始做RPTP配体屏幕，几年后，我们修改了协议，允许超过GFP的平衡器保持股票的大集合的快速分析。我们发现GFP阴性胚胎可以很容易地从GFP平衡的股票进行排序，即使有大量的产妇GFP表达（如TM3armGFP平衡器），因为胚胎进行排序可以很容易地检测到GFP表达的生活和细微的差别。我们为LAR配体的成功DF屏幕描述[5]。 使用我们当前的协议，一个单一的训​​练有素的个人可以通过屏幕5-10表型线，每天从每行的复合显微镜下检查4-7突变胚胎。选择和阵列胚胎后，大约需要一小时解剖50个胚胎。这种方法使我们能够确定突变赋予微妙，低渗透率NCE的表型，因为每行最多70 hemisegments是在高放大倍率取得浸水一个40X镜头。这种表型将很难或者不可能在染色整个安装胚胎屏幕检测，在解剖显微镜下。 我们已经定义了一个包含大约250行的表型筛选和代表约一半的基因组（ 仙等人，在准备中）DF套件。本试剂盒的发展，开始我们布卢明顿DF套件屏幕的一部分，因为它在2002-2003年RPTP配体的合成[5]需要的基因存在。在此屏幕的过程中，我们取代布卢明顿套件DFS DF / DF纯合子没有发展与较小的Dfs（CNS，因此不能为CNS配体表达筛选），最好的分子映射，即有更好的发展。 从我们的表型筛选试剂盒行的DF / DF纯合子阶段16正常的整体形态，可影响中枢神经系统，PNS的肌肉纤维，气管网，和其他很多组织发展基因的调查，系统屏幕。任何结构，表达模式，或visualizable阶段14日至16日在一个特定的抗体或绿色荧光蛋白标记的亚细胞定位模式，可以使用这个套件的表型的筛选。这意味着使用的协议，这里描述的，他/她在这个项目上的时间大约一半的人支出可以为任何需要的胚胎表型的系统屏幕在6个月到一年内所有的果蝇基因的一半。 A. 果蝇胚胎收集 1。准备"五管"鸡蛋收集室这些商会节省时间和精力，在研究大量的飞线。 <ol><li>安排500毫升锥形管，倒挂在100 × 15 mm塑料培养皿和胶水管一起使用硅橡胶粘接密封胶的底部部分。 </li><li>一旦硬化的胶水，胶水到锥形管两端的培养皿底部的一部分。五锥形管，将站内的培养皿。 </li><li>用热针，在管的孔，空气流通。 </li></ol> 2。准备鸡蛋收集板<ol><li>每100 × 15 mm塑料培养皿中倒入约葡萄琼脂凝胶（1％）7ML。凝胶的理想厚度约3-4毫米，印章每个锥形管，并提供24小时的水分。如果胶太厚，它可能会掉下来，当我们改变的板块。 </li><li>关闭凝胶板冷却后，盖，并把这些原来的塑料培养皿袋。紧密，密封袋，储存于4 ° C。 </li></ol> 3。准备苍蝇<ol><li>为了获得足够的鸡蛋容易选择的正确阶段的胚胎，我们试着放入一个跨&gt; 50处女&gt;男20例。 </li><li>如果一个交叉是进行筛选，组合的雄性和雌性果蝇，并保持在一个底部干酵母颗粒地段和一张薄薄的纸条（10 × 100毫米，对半折叠）飞食品瓶栽在中间食物。纸条将提供更多的表面积，从过多的水分，并保持苍蝇。 </li><li>至少3天，在室温下保持与苍蝇的小瓶。 </li><li>如果从一个瓶子的股票进行筛选，简单地转移到集合室苍蝇。 </li></ol> 4。传输苍蝇五桶鸡蛋收集室。 <ol><li>标签的鸡蛋收集商会每桶。 </li><li>酵母膏放在每个桶的鸡蛋收集板。 </li><li>准备20英寸长的标签磁带一起举行商会和板。折叠方便的板块变化，每年年底的10毫米。 </li><li>在飞瓶注入CO 2气体，并转移到标记桶的苍蝇。 </li><li>鸡蛋收集板室盖，并按下。 </li><li>各地的商会和板一起从中间琼脂平板开始的磁带。磁带将重叠的鸡蛋收集板。 </li></ol> 5。收集胚胎<ol><li>准备鸡蛋收集板:酵母膏放在每个桶的鸡蛋收集板。 </li><li>塔鸡蛋收集室的苍蝇和删除各地商会的磁带（紧紧握住老板）。 </li><li>再次挖掘下来的苍蝇，并更换新的旧板。 </li><li>收集胚胎在2-4小时的倾斜位置。 </li><li>更换所需时间的鸡蛋收集板。 </li></ol> 6。年龄胚胎鸡蛋收集板倒挂与湿90毫米圆滤纸覆盖的Petri板，并保持在理想的温度。 16平衡突变品系的胚胎阶段的解剖，我们通常在上午晚些时候的集合，然后孵化的胚胎R&gt; 22小时在19 ° C.对于增益的功能，使用该UAS/GAL4系统的研究，我们在下午和16小时的孵化收集胚胎在室温19 ° C。第二天，我们的胚胎转移到29 ° C培养箱激活UAS/GAL4系统为1或2小时。经过这段时间内，胚胎大多是15阶段，这允许足够的时间来表达GFP的胚胎进行排序和线路，使他们完成清扫时阶段16。 7。分期胚胎和GFP表达排序。 歧视胚胎的基因型，我们使用GFP的平衡器。我们研究下一个奥林巴斯GFP的解剖范围双棒磁带dechorionated胚胎（见下一节），我们选择年龄和GFP的表达，他们在同一时间。 A）GFP排序: <ol><li>平衡器，我们使用的第二号染色体（CyOarmGFP）驱动GFP的犰狳发起人表示，这是整个外胚层。 CyOarmGFP杂合子是橄榄绿色，CyOarmGFP纯合子是明亮的绿色。在CyoarmGFP股票，缺乏平衡器的胚胎容易辨别，因为他们几乎没有绿色荧光蛋白表达（他们看起来就像从没有GFP平衡器股票胚胎）。 </li><li>第三号染色体平衡器（TM3armGFP）进行排序更难使用，因为它有更多的母系驱动GFP表达。因此，越来越少绿的类别分为胚胎。有了一些实践，它通常是简单的排序从对方的少，更多的绿色胚胎，但它也有必要诉诸集群，并挑选出比别人更绿（TM3armGFP杂合子）偶尔传输后的胚胎琼脂平板上。否则，肯定会被一些不正确的基因型胚胎。 </li><li>的X平衡器FM7 KR - GAL4 UAS - GFP的。这是表示在组织特定的模式。有关阶段15-16排序的图案，是amnioserosa和表达在头两分，这是Bolwig的器官细胞。不幸的是，在其中一个要排序的胚胎阶段，amnioserosa衰落和Bolwig的器官尚未开始发光。因此，有必要在磁带上的胚胎，他们往往滚动，将比分的GFP仔细看，有必要诉诸他们的琼脂平板上。通常我们这样做了两次:一次后，立即将所有的胚胎从磁带板，并再次老化后的适当阶段，就在衬砌，准备解剖​​。有时候，我们也重新审视下GFP后的胚胎转移到PBS的池（见下文）范围的幻灯片，因为可视化的GFP是在这些条件下更好。然后，我们可以摧毁刚刚开始清扫前的绿色荧光蛋白的胚胎。 </li></ol> B）分段分期胚胎周围ST的主要工具。 14-16是肠道形态。肠道与下GFP范围的自体荧光发光。尝试阶段的胚胎之前，应咨询参考书或网站有胚胎的图片和图表（如Hartenstein和坎波斯，奥尔特加绿皮书），使人们了解他们看起来应该像什么。解剖的理想阶段是肠道时已划分成三个波段。在此之前，肠道出现作为一个单一的BLOB。人往往各种各样的BLOB阶段的胚胎，然后年龄，直到他们达到解剖的最佳舞台。肠道中的三波段阶段后，开始发展对角线带附近的尾巴，和然后线圈，胚胎逐渐明朗。在此期间，胚胎变得难以解剖，因为它不会粘在玻璃上滑动。如果要剖析后期16/early 17阶段的胚胎，这是必要的解剖许多胚胎和评估与坚持或不坚持的胚胎相关的肠道形态。这之间有所不同基因型以及。 B. 果蝇胚胎活夹层<ol style="font-weight:bold;"><li>胚胎dechorionation <ol style="font-weight:normal;"><li>准备幻灯片上双面胶带和葡萄板板坯</li><li>岁胚胎转移，从收集板双面胶带，用湿画笔。均匀扩散胚胎。 </li><li> Dechorionate双面胶带胚胎与迟钝解剖针轻轻滚过。胚胎后dechorionation，将继续坚持更强烈的针比其他的胚胎或绒毛膜，但没有强烈到磁带，从而卷绒毛膜顶部或到其他胚胎的胚胎，然后升降机它并将其传输到葡萄板板坯。基因分型和分期（见上面的详细说明），是在dechorionation过程转移到针。 </li><li>基因型和年龄诉诸后，将胚胎到一个较小的矩形板琼脂足够的宽度，让排队的一排10-15胚胎。在此板的dechorionated胚胎背侧对齐，对琼脂板和后向你面临结束。我们通常5-10胚胎行。 </li></ol></li><li>现场解剖准备幻灯片和转移胚胎<ol style="font-weight:normal;"><li>切一个长方形的小片，双面胶带，放在靠近一端超级Frost / PLUS的幻灯片（费舍尔科学，CAT＃12-550-15）。 </li><li>绘制一个矩形周围的磁带，用腊笔（30-40毫米长，全宽的幻灯片），因此，有〜3毫米的空间之间的蜡和磁带（超级HT子宫颈抹片<a href="http://www.rpicorp.com">笔，</a> www.rpicorp。 <a href="http://www.rpicorp.com">COM</a> ）和从葡萄板板坯的胚胎转移到磁带上，通过反相幻灯片，轻轻地降低到胚胎，使磁带接触到内衬的胚胎（下解剖范围进行） 。应该是对齐的胚胎，使后部朝向您。现在他们将在幻灯片上的背一面朝上。 </li><li>在胚胎立即加入1X PBS（Gibco公司配方），以填补蜡矩形。 </li></ol></li><li>胚胎活夹层<ol style="font-weight:normal;"><li>使用玻璃夹层针（从注射针头），切沿背中线，从胚胎后结束开始。 </li><li>捅的胚胎前结束，电梯和胚胎转移到幻灯片，直到它枝。保留在缓冲区中的胚胎。匹配的琼脂平板上的每一行的幻灯片，一个有序的胚胎。组织中的行，让他们将所有适合在幻灯片上。 </li><li>这里有各种可能夹层的序列，可用于生成圆角。人们通常做解剖自己，通过发展自己的优化程序。视频演示了一小截，是在胚胎后结束，双方彼此分开的体壁的序列。如果需要，还可以打破这种切割肠/肠连接，并取代肠，在这个时候到幻灯片（在视频，肠位移后完成）。然后，削减每侧后的脑叶，以单独的大脑身体墙壁。根据胚胎的年龄，可能还需要做出浅切下来的背中线切断体内壁之间amnioserosal的连接。 </li><li>然后，轻轻地粘贴下来到幻灯片的体壁的皮瓣，避免伸展或去除其表面的物质。最好的做法是，只允许针接触体壁背侧边缘的前部和后部的角落。不扩大这项运动轴突的背部，所以在最好的情况下，这会产生一个胚胎完全不变的ISN分行。在某些情况下的ISN会被截断，即使有最好的技术，因为胚胎并不总是单独完全沿背中线。如果做得正确，这种方法将保留在段体壁结构的T3 - A6（约）。 </li><li>胚胎上，人们可以离开肠，然后取出所有解剖胚胎办完所有的解剖。这是由肠道戳，取代了从胚胎，和伸展它打破其连接到前肠，位于脑叶下。或者，你可以从每个夹层中删除肠道作为一个完成它。随着肠道的发展有异常的突变体，它是一次删除所有的胆量，整理后的解剖，因为从肠道释放蛋黄可以更难以坚持下去后移植胚胎后，从doublestick磁带转移到幻灯片的优势。它有时是非常有用转移胚胎在幻灯片的底部开始（向你），而不是在顶部，如蛋黄往往由于针的典型动作向你传播。 </li></ol></li><li>固定和免疫/免疫组织化学<ol style="font-weight:normal;"><li>在这一点上，人们可以立即修复的胚胎，如果一个与抗体染色，或可以删除​​的PBS和替换的融合蛋白上清，如果一个人做现场的染色实验，以检测配体表达。方法和文献的补充方法部分中详细介绍了这方面的实验。 [5]。 </li><li>在这里，我们描述的固定协议，这是立即或融合蛋白染色后做。使用两个巴斯德吸液管，用5％的多聚甲醛的PBS取代的PBS（EMS特快专递，CAT＃15713 - S www.emsdiasum.com ）。在修复方案交易所三次，其中包括使用〜6毫升的修复方案，因为巴斯德吸管持有1.5-2毫升的解决方案。修正为45&#39;。 </li><li>删除修复方案，取代用PBS（3变化），然后与PBT（PBS + 0.1％海卫X - 100 + 1毫克/毫升分数V BSA; 3变化），然后将其替换〜200μL块解决方案（PBT PBT + 5 ％热处理正常山羊血清），然后与所需块解决方案的主要抗体。这是非常重要不允许半月板接触，特别是胚胎，而他们在PBS的，因为这会破坏了解剖。添加洗衣粉后的胚胎变得不那么敏感的半月板。因此，可以取代块小费幻灯片和印迹的解决方案，通过触摸一个Kimwipe简要蜡封闭的区域，角落最的PBT。同样的方法可用于抗人体的解决方案，以取代块。这种解决方案，最大限度地减少结转。 </li><li>孵育过夜在4 ° C，然后洗3X在一个托盘上摇摆平台与PBT（&gt; 15分钟/洗）（请确保有足够的PBT使幻灯片始终是完全沉浸在摇摆），reblock如上所述，加200μL二次抗体，孵育2小时。在室温下，与PBT rewash。如果根据解剖范围内的GFP所需的检查（如果使用绿色荧光）染色。 </li><li>洗净，用PBS（5&#39;）的2倍，再加入500μL70％glycerol/1X PBS（甘油35毫升，5毫升10X PBS，和10毫升的水混合在50毫升管），并明确在一夜之间4 ° C （或在室温下为2小时）。取出甘油放在一个＃1盖玻片。 </li></ol></li></ol>

<ol>
	<li>Patel, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+subsets+and+other+cell+types+in+whole-mount+Drosophila+embryos+and+larvae+using+antibody+probes.">Imaging neuronal subsets and other cell types in whole-mount Drosophila embryos and larvae using antibody probes.</a> Methods Cell Biol. 44, 445-487 (1994).</li><li>Desai, C. J., Gindhart, J. G., Goldstein, L. S., Zinn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+tyrosine+phosphatases+are+required+for+motor+axon+guidance+in+the+Drosophila+embryo.">Receptor tyrosine phosphatases are required for motor axon guidance in the Drosophila embryo.</a> Cell. 84, 599-609 (1996).</li><li>Sun, Q., Schindelholz, B., Knirr, M., Schmid, A., Zinn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+genetic+interactions+among+four+receptor+tyrosine+phosphatases+regulate+axon+guidance+in+Drosophila.">Complex genetic interactions among four receptor tyrosine phosphatases regulate axon guidance in Drosophila.</a> Molecular and cellular neurosciences. 17, 274-291 (2001).</li><li>Schmid, A., Schindelholz, B., Zinn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+RNAi:+a+method+for+evaluating+the+functions+of+gene+families+in+Drosophila.">Combinatorial RNAi: a method for evaluating the functions of gene families in Drosophila.</a> Trends in neurosciences. 25, 71-74 (2002).</li><li>Fox, A. N., Zinn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+heparan+sulfate+proteoglycan+syndecan+is+an+in+vivo+ligand+for+the+Drosophila+LAR+receptor+tyrosine+phosphatase.">The heparan sulfate proteoglycan syndecan is an in vivo ligand for the Drosophila LAR receptor tyrosine phosphatase.</a> Curr Biol. 15, 1701-1711 (2005).</li></ol>

我们希望这个视频演示，显示了我们的活胚胎解剖和任何人有一些在果蝇的遗传学和胚胎学的经验，他们现在可以很容易执行的足够详细的染色方法。当然，将仍然需要相当的实践，主要用于解剖自己。根据我们的经验，每个人学习生活的清扫方法，将创建一个微妙的不同剥离协议，允许他/她最快速的生成高质量的摆着解剖。这个过程需要全面发展的个月。然而，大多数人经过几个星期的...

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<td>Borosilicate Tubing With Filament </td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>BF120-60-10</td>
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<td>Narishige model PC-10 needle puller</td>
<td>&nbsp;</td>
<td>Narishige</td>
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<td>Tubes pulled using a heater level of 58.9</td>
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live dissection of drosophila embryos: streamlined methods for screening mutant collections by antibody staining

果蝇胚胎胚胎发育的阶段，14和17之间，可以很容易地解剖产生"鱼片"的准备工作。在这些准备工作，中枢神经系统运行的中间，是由身体墙壁两侧。许多不同表型已使用这种制剂的研究。鱼片在大多数情况下，所产生的抗体染色的固定安装整个胚胎的夹层。这些"固定的解剖"，但有一些缺点。他们耗费时间来执行，也很难在保持了GFP平衡器染色体突变的股票进行排序的突变体（GFP阴性）胚胎。自2002年以来，本集团一直在进行缺乏症和异位表达屏幕确定为孤儿受体的配体。为了做到这一点，我们开发的精简协议活胚胎剥离和抗体染色包含数百个平衡线路的集合。我们得出的结论，这是相当多的效率比固定清扫现场清扫研究股票的大集合的表型。使用这里描述的协议，单一的训练有素的个体可以通过屏幕10号线每天的表型，从每行的复合显微镜下检查4-7突变胚胎。这使得查明赋予微妙，低外显的表型的基因突变，在高放大倍率拿下了一个40倍的水浸泡镜片，因为每行最多70 hemisegments。

Watch this Scientific Journal Video about Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining at JoVE.com

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

我们描述了一个用于生成特定基因型的果蝇胚胎的"鱼片"筹备工作的精简的协议。此协议允许多种遗传筛选的高效执行。它也可以让优秀的中后期的胚胎结构的可视化。

现场剖析果蝇胚胎抗体染色筛选突变体集合的简化方法

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

live-dissection-drosophila-embryos-streamlined-methods-for-screening

Research

JoVE Journal

Biology

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

14.2K 次观看.  California Institute of Technology. 我们描述了一个用于生成特定基因型的果蝇胚胎的"鱼片"筹备工作的精简的协议。此协议允许多种遗传筛选的高效执行。它也可以让优秀的中后期的胚胎结构的可视化。

视频: Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Dissection of Drosophila Ovaries

剖析果蝇卵巢

科研

生物学

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

6.5 DPC小鼠胚胎剖析

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

整装在鸡的抗体染色方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

果蝇幼虫NMJ夹层

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

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 Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

果蝇聚乙烯南瓜抗体标记染色体的制备

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

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 ...

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 ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

夹层和染色果蝇幼虫卵巢

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...