我们感谢露丝雷曼Dorthea戈特谁好心提供瓦萨和交通拥堵的抗体。我们要感谢布卢明顿库存中心在美国印第安纳大学维护提供股票和发展研究杂交瘤细胞银行NICHD的赞助下开发和爱荷华大学维护。我们感谢Wawersik实验室全体成员的意见和支持。这个工作是由梦露学者项目资助（以自动对焦和LB）和美国国家科学基金会授予IOS0823151（以兆瓦）。

1，准备集合笼<ol><li>麻醉年轻，肥沃的苍蝇与CO 2。转让麻醉苍蝇笼子里。为了获得最佳的收益率，用100-120成蝇从2-7日龄以4:1的比例女性为男性。让苍蝇适当的适应期，〜24小时之前获得的样品进行固定。如果笼成立了处女的女性交配雄性，一个用36 - 48小时的适应期。 </li><li>在笼的开放端，将事先准备好的苹果汁琼脂平板上用酵母膏中的中心在保持器的开口部的硬币大小的滴。然后，用固定带。密封的苹果汁琼脂平板上，并用封口膜笼之间的交界处。 </li><li>放置笼中以苹果汁琼脂板作为基材的次级容器，并允许如由实验确定的苍蝇重现在确定的温度下在适当的时间周期。 注：样品采集时间无线会有所不同。例如，收集后期胚胎/初的L1幼虫时（17 - 24小时），使苍蝇，在25℃，在25℃放置在苹果汁琼脂平板鸡蛋7小时前样品的老化17小时。类似地，对于中后期第一龄期幼虫的集合（36 - 48小时），使苍蝇，在25℃，在25℃产卵12小时之前的样品老化36小时。 </li><li>一旦时间周期完成后，点击桌子上的笼子里，让苍蝇落离苹果汁琼脂平板无需麻醉。用新鲜含一滴酵母膏迅速更换使用的板材。保证新鲜板用胶带和封口膜和存储与苹果汁琼脂平板为基础的笼子。 </li><li>小心地从旧板酵母滴用金属铲。所使用的苹果汁板盖的地方，并允许放置胚胎的年龄，如果实验需要。 注意：在老化样品中，板可以被存储在25℃下，除非较高的温度是experimentaLLY要求。如何变老的胚胎收集上面描述的例子（见注步1.3）。除去样品老化前酵母膏的被执行以防止幼虫从爬行到酵母和分手琼脂下方。剩下的苹果汁琼脂和酵母膏残渣提供养分的幼虫的生长。而胚胎直接在酵母膏敷设是通过酵母膏除去丢失，这种损失是优选的酵母和琼脂残余的样品，可以减少染色效率。应选择其中一种不以除去酵母膏，酵母可与磷酸盐缓冲液的Triton X-100（PBTx）液化，用漆刷混合，然后从用细胞过滤网之前，固定样品中除去。 </li></ol> 2，固定<ol><li>一旦胚放置到苹果汁琼脂平板上有年龄到所需的时间点，用小画笔润湿的磷酸盐缓冲液的Triton X-100（PBTx）溶液小心地从第除去样品e盘。 注：以前的幼虫是移动的，并且抓取到盖内。此示例同样可以通过去除用画笔来收集。 </li><li>擦拭针对细胞过滤器的内部面对脊样本载货画笔。然后，用水冲洗过滤器的壁，并与含有PBTx溶液喷射瓶的画笔。将培养皿盖上的滤网下方捕捉PBTx解决方案。 </li><li>之后所有的样品已经被转移到过滤器，倾足够PBTx进入过滤器，以提高样品脱网。然后，抬起过滤，倒入培养皿中的内容变成液体废物容器。 </li><li>重复步骤2.3三次以除去酵母和飞可能已随着样品转移废物。 </li><li>浇足50％的漂白剂（次氯酸钠）/水（DDH 2 O） 的溶液入过滤，以提高幼虫脱网。让幼虫坐在溶液5分钟。然后，抬起过滤，倾吨他在液体废物容器中的陪替氏培养皿的内容。 注意：漂白剂仅需要在0岁胚胎样 - 为了除去绒毛膜22小时。漂白剂的较旧的样本应用程序可以省略，但其夹杂物是无害。如果省略的需要，更换漂白剂在此步骤PBTx。稀释的漂白水应在溶液的配制24小时使用。 </li><li>浇足PBTx入过滤器，以提高样本断网与PBTx过滤洗涤样品。允许样品坐在溶液3分钟。然后，抬起过滤，倒入培养皿中的内容变成液体废物容器。 </li><li>重复执行步骤2.6的五倍。 </li><li>民建联的细胞过滤干燥的底部，然后转移到样品含有1.75毫升用画笔PEMS系统解决方案，闪烁瓶中。 </li><li>工作在一个通风良好的通风橱中，加入250μl的37％的甲醛和8ml试剂级庚烷到闪烁瓶中。 <br/&gt;注意：甲醛和庚烷是危险的。为安全起见，继续在通风橱工作，并戴上适当的手套步骤2.9 - 3.3。 </li><li>允许小瓶以摇动以200rpm或类似的中等速度持续20分钟。 </li><li>加入10毫升甲醇，以闪烁小瓶的不除去先前的水相，并且允许小瓶以剧烈摇晃以500rpm持续1分钟。 注：甲醇是危险的。继续在通风橱工作，并配戴合适的手套。 </li><li>从摇瓶中取出，并立即倒入内容放到一个细胞过滤过的引擎盖液体废物容器。加入额外的甲醇的小瓶中，通过在必要时的细胞过滤器运行，以确保所有的样品已经被移除。 注：细胞滤网可能会耗尽缓慢。为了解决这个问题，触摸实验室组织的细胞过滤器的底部，以便更快速地绘制通过过滤的溶液。甲醇，甲醛和庚烷应存储在适当的废弃物容器，直至妥善处置按照机构的指导方针。 </li><li>细胞过滤器的使用实验室的组织干的底部，然后用画笔样品中的过滤器捕获转移到含0.5毫升甲醇1.5 ml离心管中。 注：如果有多个闪烁瓶正在使用，完成步骤2.12和2.13一个小瓶在同一时间，以防止样品在干细胞过滤器。 </li><li>一旦样品已经被添加到微量离心管中，除去甲醇用移液管。确保样品已经沉降到管底。 </li><li>通过将大约为0.5毫升甲醇中，以管冲洗在微量离心管中的样品。等待样品来解决，然后用吸管除去甲醇。 </li><li>重复步骤15三次。 </li><li>加入0.5毫升甲醇中的管，并且然后存储在-20℃下冷冻以备后用。 </li></ol> 3，补液和样本的免疫染色的制备<ol><li>从离心管中用吸管除去甲醇，留下在管中的样品。商店甲醇废弃物的适当的废物容器中，直到妥善处理时间。 注意：为了提高超声处理的效率，可以使用不超过3毫米的实例的在1.5 ml离心管的底部结算。过量样品可以开始上述补液步骤之前被转移到单独的试管用移液管。切割枪头用干净的刀片可用于防止移液管尖的堵塞。 </li><li>加入1.0毫升的50％的甲醇/磷酸缓冲液吐温（PBTw）溶液于管和岩石3分钟。 </li><li>除去甲醇溶液，以适当的废弃物容器中，用1.0微升PBTw的冲洗两次。每次冲洗后，让样品图纸掉PBTw用吸管之前结清。 </li><li>加入1.0毫升牛血清白蛋白/磷酸盐缓冲吐温（BBTw）的混小瓶和岩石。后摇臂3分钟，让SAMP乐定居并删除BBTw用吸管。 </li><li>重复步骤3.4两次照顾以除去尽可能多的BBTw尽可能没有在最后洗涤后丢失样本。 </li><li>加入0.5毫升BBTw到管，并放置在管上的冰。 </li></ol> 4，超声样品<ol><li>将超声波仪，以最大振幅10％，并指定2秒不变超声波运行时间。 注意：这些设置是特定于在材料和设备表所示的超声仪，并可能需要优化不同sonicators。 </li><li>前样品的超声处理，通过将探针针尖到50ml锥形管中填充有离子交换水和运行超声波仪，以清洁探头清洗超声波仪探针。干式超声仪探头运行后的实验室组织。 </li><li>浸入探头到含有样品的离心管中，以便它被定位大约3至4mm的样品上面，开始超声处理。 </li><li>卸下米icrocentrifuge管，并放置在冰上至少30秒，从超声处理散热，并允许样品定居在微量离心管的底部。 </li><li>重复步骤4.3和4.4在需要的基础上样品的年龄正在处理中。 注：为获得最佳超声样品体积，胚胎年龄小于17小时不需要超声，但那些17至24小时（台17 /早期L1）需要两个超声处理。幼虫在24 - 36（早/中L1），36 - 48（中/后期-L1），48 - 60（早/中二级），60 - 72（中/晚L2），72 - 84（早L3），84 - 96（早期/中期L3），96 - 108（中/晚L3）小时需要5，9，11，13，15，18，和22超声处理。见进一步阐述超声时间的讨论。 </li><li>用1.0mL冲洗BBTw两次。每次漂洗后使样品拉伸断BBTw用移液管之前解决。 </li><li>加入1.0毫升BBTw到小瓶和岩石。后在摇臂3分钟，使样品沉淀，除去BBTw用吸管。&lt;/ LI&gt; <li>重复步骤4.7的两倍。 </li></ol> 5，免疫染色<ol><li>通过添加5％正常血清稀释于BBTw到微量离心管中阻止样品。摇1小时后，取出块。 注意：使用正常血清从其中产生的次级抗体的物种。 </li><li>稀释的初级抗体，以适当的在5％正常血清/ BBTw溶液工作浓度。 </li><li>初级抗体溶液的O /岩石样本n在4℃或至少3小时，在室温（约22℃。 注：抗体孵育时间和温度可以变化。 O 2 / N孵育在4℃下，或3小时，在室温通常是足够的。然而，两天潜伏期可以提高染色质量，特别是年龄较大的样本，或当低亲和力的第一抗体被使用。较长的孵育，通常进行的，在4℃。类似的考虑，必须使用二次抗体（见5.10）时进行。 </li><li>使样品稳定到离心管的底部。抽出一抗用吸管。如果需要保存的抗体以重复利用。 </li><li>用1.0mL BBTw的冲洗两次。每次冲洗后，使样品被抽走BBTw用吸管之前结清。 </li><li>加入1.0毫升BBTw到小瓶和岩石。后在摇臂3分钟，使样品沉淀，除去BBTw用吸管。 </li><li>重复执行步骤5.6的五倍。 </li><li>通过添加5％正常血清/ BBTw溶液的微量离心管块样品。摇动30分钟至1小时后除去块。 注意：不需要这个额外的封闭步骤，但可以提高染色质量的特异性抗体。 </li><li>稀释二级抗体，以适当的在5％正常血清/ BBTw溶液工作浓度。 </li><li>包裹的微离心管中的铝箔，以减少衰落是由于光照射。中，在4℃下进行12至48小时的二级抗体溶液或至少3小时，在室温（约22℃的岩石样品。 </li><li>使样品沉淀到离心管的底部。抽出的第二抗体与吸管。 </li><li>用1.0mL PBTw的冲洗两次。每次冲洗后，使样品被画掉PBTw用吸管之前结清。 </li><li>加入1.0毫升PBTw到小瓶和岩石。 5分钟后，在摇臂，使样品沉淀，除去PBTw用吸管。 </li><li>重复步骤5.13三次。 </li><li>可选：添加DAPI稀释1：1,000 PBTw。岩石样品包在铝箔3分钟;然后取出用吸管DAPI的解决方案。冲洗五次1.0毫升PBTw，让样品用吸管取出PBTw前结清。 </li><li>加80 - 100微升1,4 - 二氮杂二环[2.2.2]辛烷（DABCO）溶液至离心管中并储存在-20℃。 注：另甘油类抗褪色剂可以被取代的。 </li></ol> 6，分析<ol><li>安装样品在载玻片上之前，添加一个额外的5 - 10微升DABCO / P-啉的ylenediamine（PPD）抗褪色溶液至离心管中。 注：样品可以被添加到载玻片没有清扫。添加到幻灯片的样本量随盖玻片大小。当移取样品上滑动时，移液管尖端可以用剃刀刀片，以防止样品的剪切被切断。它往往是有益的在线样品在便于分析行。可以不需要添加的PPD作为附加抗淬灭剂，但可以防止光致漂白，如果样本被存储长期。 </li><li>轻轻将盖玻片上的样品。然后，在安全的地方盖玻片使用指甲油。 </li><li>视图上的样品用荧光显微镜。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+cell+rearrangement+and+cell+recruitment+in+Drosophila+ovary+morphogenesis+and+the+requirement+of+bric+a+brac.">Mechanisms of cell rearrangement and cell recruitment in Drosophila ovary morphogenesis and the requirement of bric a brac.</a> Development. 121, 173-187 (1995).</li><li>Gancz, D., Lengil, T., Gilboa, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+regulation+of+niche+and+stem+cell+precursors+by+hormonal+signaling.">Coordinated regulation of niche and stem cell precursors by hormonal signaling.</a> PLoS Biol. 9, e1001202 (2011).</li><li>Matsuoka, S., Hiromi, Y., Asaoka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Egfr+signaling+controls+the+size+of+the+stem+cell+precursor+pool+in+the+Drosophila+ovary.">Egfr signaling controls the size of the stem cell precursor pool in the Drosophila ovary.</a> Mech Dev. 130, 241-253 (2013).</li><li>Song, X., Zhu, C. H., Doan, C., Xie, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+stem+cells+anchored+by+adherens+junctions+in+the+Drosophila+ovary+niches.">Germline stem cells anchored by adherens junctions in the Drosophila ovary niches.</a> Science. 296, 1855-1857 (2002).</li><li>Homem, C. C., Knoblich, 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=Drosophila.+neuroblasts:+a+model+for+stem+cell+biology.">Drosophila. neuroblasts: a model for stem cell biology.</a> Development. 139, 4297-4310 (2012).</li><li>Urbach, R., Technau, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroblast+formation+and+patterning+during+early+brain+development+in+Drosophila.">Neuroblast formation and patterning during early brain development in Drosophila.</a> BioEssays. 26, 739-751 (2004).</li><li>Bello, B., Reichert, H., Hirth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brain+tumor+gene+negatively+regulates+neural+progenitor+cell+proliferation+in+the+larval+central+brain+of+Drosophila.">The brain tumor gene negatively regulates neural progenitor cell proliferation in the larval central brain of Drosophila.</a> Development. 133, 2639-2648 (2006).</li><li>Lee, C. Y., Wilkinson, B. D., Siegrist, S. E., Wharton, R. P., Doe, C. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brat+is+a+Miranda+cargo+protein+that+promotes+neuronal+differentiation+and+inhibits+neuroblast+self-renewal.">Brat is a Miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal.</a> Dev Cell. 10, 441-449 (2006).</li><li>Betschinger, J., Mechtler, K., Knoblich, 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=Asymmetric+segregation+of+the+tumor+suppressor+brat+regulates+self-renewal+in+Drosophila+neural+stem+cells.">Asymmetric segregation of the tumor suppressor brat regulates self-renewal in Drosophila neural stem cells.</a> Cell. 124, 1241-1253 (2006).</li><li>Pereanu, W., Shy, D., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+and+proliferation+of+the+larval+brain+glia+in+Drosophila.">Morphogenesis and proliferation of the larval brain glia in Drosophila.</a> Dev Biol. 283, 191-203 (2005).</li><li>Moraru, M. M., Egger, B., Bao, D. B., Sprecher, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cell+identity,+morphology,+apoptosis+and+mitotic+activity+in+a+primary+neural+cell+culture+system+in+Drosophila.">Analysis of cell identity, morphology, apoptosis and mitotic activity in a primary neural cell culture system in Drosophila.</a> Neural Dev. 7, 14 (2012).</li></ol>

The authors have nothing to disclose.

这个协议提供了一种方法来成功地免疫组织化学染色的目标果蝇胚胎和幼虫组织原位 ，从而省去了清扫。按照现有协议进行染色早期胚胎1,2,3，绒毛膜膜是用50％的漂白剂（次氯酸钠）除去。样品被固定在甲醛和甲醇。因为幼虫角质层导致老年人样品浮起，将整个样品然后通过细胞过滤器传递到保证幼虫保留。样本被存储，如果需要的话，在化学级甲醇。补液后，正确实施超...

果蝇胚胎和幼虫为研究发育过程中的许多器官和组织一个很好的典范。各个单元的成像是在这些研究中常常需要以确定在哪些细胞发育的复杂的环境。细胞在组织中的可视化可以通过免疫染色来完成。以及描述的免疫染色协议果蝇胚胎组织&lt;后17小时产蛋（AEL）1-3存在。然而，保护表皮的形式朝向胚的末端，以防止有效抗体渗透。因此，这些免疫染色方案是低效的中后期胚胎组织的分析和在幼虫发育（ 第一龄（L1）， 第二龄（L2）和第三龄（L3））的后续阶段。这种低效率施加了障碍，我们在这延长的发展阶段4所发生的动态过程的理解。 TISSUE夹层是一种广泛使用的技术来绕过这一障碍5-7。然而，夹层可以证明效率低下。提取可以通过难以定位或分离胚胎和幼虫组织缠身。此外，物理去除目标组织可以通过破裂它们或通过不提取它们以其整体造成损坏。 超声是采用声波干扰分子间相互作用的方法。它已被用于以免疫染色显影神经细胞类型6打乱果蝇幼虫角质层的完整性。这个协议已经适应免疫染色后期胚胎和幼虫性腺，它可以小到50微米的直径8-10。通过这样的研究中，雄性生殖系干细胞（GSC）利基形成过程的特点在后期果蝇胚胎8-10和机制调节干细胞的发育和DIFferentiation晚期胚胎性腺和幼虫已经阐明9-12。因此，超声处理提供了一种有效的替代组织剥离，可能是由于组织大小困难。此外，它使果蝇组织中的原位免疫染色，而使整个有机体的范围内的细胞和保持在原位的形态。在这里，我们描述了通过早期/中期三级组织原位一步一步的协议中晚期胚胎荧光免疫染色。 果蝇性腺和神经组织的分析显示，在代表性的成果来证明这个协议的效力。此外，这种免疫方案可适于分析其他果蝇组织以及组织中其他生物具有外表皮。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1: Reagents and Buffers</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Triton X-100 (PBTx)</td>
			<td>For 5 L: 500 mL PBS 10X 4.45 L ddH2O 50 mL Triton 10%</td>
			<td>Store at room temperature.</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline 10x (PBS 10X)</td>
			<td>For 1 L in dH2O: 80 g NaCl 2 g KCl 14.4g Na2HPO4 2.4 g KH2PO4</td>
			<td>Add components and fill to appropriate volume. Store at 4 degrees C.</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton 10%</td>
			<td>For 50 mL: 5 mL of Triton 5 mL of PBS 10X 45 mL of ddH2O</td>
			<td>Rock to mix. Store at room temperature.</td>
			<td></td>
		</tr>
		<tr>
			<td>PEMS</td>
			<td>0.1 M Pipes (pH 6.9) 2.0 mM MgSO4 1.0 mM EGTA</td>
			<td>Store at room temperature.</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipes</td>
			<td>For a 400ml of a 0.25 M solution (pH 6.9): 30.24 g Pipes dH20 NaOH</td>
			<td>Dissolve Pipes in 300 mL dH2Oand then adjust to pH 6.9 with NaOH. Bring the total volume to 400 mL with dH2O and autoclave. Store at room temperature.</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>37% formaldehyde by weight in methanol</td>
			<td>Store at room temperature. Store formaldehyde, heptane, and methanol waste mixture in a tightly sealed container in fume hood before disposal as per institutional guidelines.</td>
			<td></td>
		</tr>
		<tr>
			<td>Heptane</td>
			<td>n-Heptane</td>
			<td>CAS 142-82-5. Store at room temperature. Store formaldehyde, heptane, and methanol waste mixture in a tightly sealed container in fume hood before disposal as per institutional guidelines.</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Methanol</td>
			<td>CAS 67-56-1. Store at room temperature. Store formaldehyde, heptane, and methanol waste mixture in a tightly sealed container in fume hood before disposal as per institutional guidelines.</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Tween (PBTw)</td>
			<td>To make 1 L: 100 mL PBS 10X 890 mL ddH2O 10 mL Tween 10%</td>
			<td>Filter sterilize after adding all components. Store at 4 degrees C.</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 10%</td>
			<td>For 50 mL: 5 mL Tween 5 mL of PBS 10X 40 mL of ddH2O</td>
			<td>Rock to mix. Store at room temperature.</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin/Phosphate Buffer Tween (BBTw)</td>
			<td>To make 1 L: 100 mL PBS 10X 890 mL ddH2O 10 mL Tween 10% 1 g Bovine Serum Albumin (BSA)</td>
			<td>Add BSA then sterilize using a 0.2 micrometer vacuum filter unit. Store at 4 degrees C.</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum (NGS)</td>
			<td>To make 10 mL: Normal Goat Serum (Jackson ImmunoResearch Laboratories Code: 005-000-121) 10 mL ddH2O</td>
			<td>Add ddH2O to vial of NGS and sterilize using a 0.2 micrometer syrninge filter. Store aliquots at -20 degrees C.</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-diazabicyclo[2.2.2]octane (DABCO)</td>
			<td>To make 100 mL: 25 mL ddH2O 1 mL Tris HCl (1M, pH 7.5) 2.5 g of DABCO solid (CAS: 281-57-9) 3.5 mL 6N HCl 250 uL 10N NaOH 70 mL glycerol</td>
			<td>In 250 mL beaker with stir bar, add ddH2O, Tris HCl and DABCO. Stir and then add 6N HCl, 10 N NaOH, and glycerol. Then add 10NaOH dropwise until solution reaches pH 7.5. Aliquot. Store aliquots at -20 degrees C.</td>
			<td></td>
		</tr>
		<tr>
			<td>DABCO + p-phenylenediamine (PPD) Solution</td>
			<td>1.765 mL NaHCO3 0.353 Na2CO3 0.02 g PPD (CAS: 106-50-3)</td>
			<td>Dissolve PPD in NaHCO3 and NaCO3 solution. Add 60 uL of PPD solution to 500 uL of DABCO. Store aliquots at -20 degrees C.</td>
			<td></td>
		</tr>
		<tr>
			<td>Apple juice plates</td>
			<td>To make ~200 plates: 45 g agar (CAS#9002-18-0) 45 g granulated sugar (store bought) 500 mL Apple juice (store bought) 15 mL Tegosept 10% 1.5 mL ddH2O</td>
			<td>Add agar to ddH2O in 4L flask then autoclave for 30 minuntes. Mix apple juice and sugar on heated stir plate. Gradually add apple juice mixture to autoclaved agar. Mix on heated stir plate then aliquot 10mL volumes into 35 mm petri dishes and let stand at room temperature to solidfy. Store at 4 degrees C.</td>
			<td></td>
		</tr>
		<tr>
			<td>Tegosept 10%</td>
			<td>To make 100mL: 10g Tegosept 100 mL Ethanol</td>
			<td>Store aliquots at -20 degrees C</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast paste</td>
			<td>~50 g Dry Active Yeast</td>
			<td>Gradually add ddH2O to beaker containing yeast while stirring until paste-like consistency reached. Store at 4 degrees C.</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2: Staining Materials</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>1:1000</td>
			<td>Invitrogen</td>
			<td>D3571 //// Stock at 5mg/mL</td>
		</tr>
		<tr>
			<td>rabbit anti-Vasa</td>
			<td>1:250</td>
			<td></td>
			<td>A gift from Ruth Lehmann</td>
		</tr>
		<tr>
			<td>mouse anti-Fasciclin III</td>
			<td>1:10</td>
			<td>Developmental Studies Hybridoma Bank (DSHB)</td>
			<td>7G10</td>
		</tr>
		<tr>
			<td>mouse anti-1B1</td>
			<td>1:4</td>
			<td>Developmental Studies Hybridoma Bank (DSHB)</td>
			<td>1B1</td>
		</tr>
		<tr>
			<td>guniea pig anti-Traffic Jam</td>
			<td>1:2500</td>
			<td></td>
			<td>A gift from Dorthea Godt (Li et al, 2003)</td>
		</tr>
		<tr>
			<td>mouse anti-Prospero</td>
			<td>1:10</td>
			<td>Developmental Studies Hybridoma Bank (DSHB)</td>
			<td>Prospero MR1A</td>
		</tr>
		<tr>
			<td>rat anti-Elav</td>
			<td>1:30</td>
			<td>Developmental Studies Hybridoma Bank (DSHB)</td>
			<td>Rat-elav 7EBA10 anti-elav</td>
		</tr>
		<tr>
			<td>mouse anti-Repo</td>
			<td>1:10</td>
			<td>Developmental Studies Hybridoma Bank (DSHB)</td>
			<td>8D12 anti-Repo</td>
		</tr>
		<tr>
			<td>goat anti-rabbit Alexa546</td>
			<td>1:500</td>
			<td>Invitrogen</td>
			<td>A11010</td>
		</tr>
		<tr>
			<td>goat anti-mouse Alexa488</td>
			<td>1:500</td>
			<td>Invitrogen</td>
			<td>A11029</td>
		</tr>
		<tr>
			<td>goat anti-guniea pig Alexa633</td>
			<td>1:500</td>
			<td>Invitrogen</td>
			<td>A21105</td>
		</tr>
		<tr>
			<td>goat anti-rat Alexa488</td>
			<td>1:500</td>
			<td>Invitrogen</td>
			<td>A11006</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 3: Materials and Equipment</td>
		</tr>
		<tr>
			<td>Fly Cages</td>
			<td>Hand-made; Genesee Scientific Corporation</td>
			<td>Not applicable; Bottles: 32-130; Pre-made cage: 59-101</td>
			<td>Made by cutting clear cast acrylic tubing (1 3/4 inch in diameter) into 4 inch tall segments with a compound miter saw at 400 rpm. Ultrafine stainless steel screening (was attached to one end of the tub with acrylic compund glue. An alternate method using an empty fly food bottle can be found in Drosophila Protocols ISBN 0-87969-584-4. Cages may also be purchased from the Genesee Scientific Corporation.</td>
		</tr>
		<tr>
			<td>Sonicator: Branson 250 Digital Sonifier</td>
			<td>Branson</td>
			<td>Model: Branson Digital Sonifier 250</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator Probe</td>
			<td>Branson</td>
			<td>Model #: 102C (CE)</td>
			<td>EDP: 101-135-066; S/N: OBU06064658</td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Nalgene</td>
			<td>190-25-20</td>
			<td>0.2 micrometer cellulose, acetate membrane filter</td>
		</tr>
		<tr>
			<td>Imaging system: Spinning disc confocal microscope with multichromatic light source, digital CCD camera, and imaging software</td>
			<td>Microscope: Olympus Light source: Lumen Dynamics Camera: Q-Imaging Imaging Software: Intelligent Imaging Inc.</td>
			<td>Microscope: BX51 equipped with DSU spinning disc Light source: X-Cite 120Q Camera: RETIGA-SRV Imaging Software: Slidebook 5.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum filter unit</td>
			<td>Nalgene</td>
			<td>450-0020</td>
			<td>0.2 micrometer cellulose nitrate membrane filter</td>
		</tr>
	</tbody>
</table>

sonication-facilitated immunofluorescence staining of late-stage embryonic and larval drosophila tissues in situ

在果蝇进行研究果蝇胚胎和幼虫提供重要的洞察，如细 ​​胞命运规范和器官发育过程。免疫允许开发组织和器官的可视化。然而，保护角质层形成于胚胎发育的最后阻止抗体渗透到后期的胚胎和幼虫。虽然之前的免疫清扫经常用来分析果蝇幼虫组织，它证明效率低下的一些分析，因为小组织可能难以定位和分离。超声提供了果蝇幼虫的免疫方案替代清扫。它允许大量的后期胚胎和幼虫的快捷，同时处理和维护现场形态。固定在甲醛后，将样品进行超声处理。样品然后进行免疫染色与抗原特异性初级蚂蚁ibodies和荧光标记的第二抗体，通过荧光显微镜可视化靶细胞类型和特定的蛋白质。在超声处理的过程中，超声处理探针的样品上面，以及超声处理的持续时间和强度，正确放置是关键的。可能需要高品质的污渍产生额外稍做修改标准的免疫染色的协议。对于抗体具有低的信噪比，更长的孵育时间通常是必要的。至于概念，这种超声促进的协议的证明，我们将展示三种组织类型（睾丸，卵巢，和神经组织）免疫组化染色，在一系列的发展阶段。

为了证明在晚期胚胎和原位幼虫组织分析超声系免疫的功效，野生型胚胎和幼虫处理用于免疫睾丸，卵巢，和神经组织。样品通过共聚焦显微镜成像和代表性结果示（ 图1和图2）。结果表明，所描述的协议是有效的用于可视化的形态特征以及各个细胞在原位时果蝇发育的晚期胚胎通过早期/中期L3阶段。 结果从睾丸免疫组织?...

Watch this Scientific Journal Video about Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ at JoVE.com

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.

后期阶段的胚胎和幼虫的超声，促进免疫荧光染色<em&gt;果蝇</em&gt;组织<em&gt;原位</em

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and ...

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Biology

Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

12.5K 次观看.  College of William & Mary. Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae. 

视频: Sonication-facilitated Immunofluorescence Staining of Late-stage Embryonic and Larval Drosophila Tissues In Situ

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

在果蝇幼虫中枢神经系统的剖析

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

科研

生物学

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

从晚期果蝇蛹的大脑原发性神经文化

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

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 ...

Drosophila Larval NMJ Immunohistochemistry

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 31 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. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

果蝇幼虫NMJ免疫

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 ...

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

斑马鱼胚胎眼组织显微切割的

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

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 ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

从解离分析准备胚胎和幼体斑马鱼骨肌纤维

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

微RNA<em&gt;原位</em&gt;杂交的福尔马林固定的肾组织

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

幼虫活动的测量<em&gt;果蝇</em&gt;活动监视器

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...