我们感谢赖科·凯泽和Tomotsune Ameku他们为这项工作提供技术支持。我们也感谢凯·托，奥尔加·亚历克西科，明子江东，马赛基·米拉，布鲁明顿果蝇库存中心，京都库存中心（DGRC），以及发展研究杂交瘤细胞银行股票和试剂。这项工作是由日本学术振兴会KAKENHI授权号16K20945，内藤基金会，和井上学研究奖授予YSN支持;并通过文部科学省从格兰特KAKENHI数16H04792一笔赠款，RN。

注:协议的总体方案示于图1。 1.幼虫环压盖的解剖（RG） 注:在D中。 黑腹果蝇 ，属于cyclorrhaphous双翅目，在PG是内复合内分泌器官称为环腺（RG， 图2D）。既然是不可行的是，PG被手术从其它类型的细胞（稍后讨论）的分离，一个实际的目标是通过解剖分离完整的，未损坏的RG。 <ol><li> 幼虫在适当的发育阶段的准备 注意:要同步果蝇幼虫的发育阶段，有必要窄时间窗内收集鸡蛋，并在适当的时间，以收集幼虫。 <ol><li>在25℃下收集鸡蛋用于在葡萄果汁琼脂平板2个小时。 </li><li>收集刚孵出1 <sup&gt;用镊子在解剖显微镜下第一龄幼虫，并将它们传送到包含捣碎标准酵母/玉米面飞食品小瓶。 注:幼虫的年龄由下式表示，"产卵（HAEL）小时后"，"孵化后小时（HAH）"，或"L3蜕皮（hAL3E）小时后"。 </li><li>在适当的时间点，收集与一次性塑料循环上演幼虫避免不必要的伤害。为了最小化在第三龄幼虫的发育进展的差，收集在70 HAEL（48 HAH）第2 次龄幼虫，并允许他们在2周小时的时间间隔蜕皮。然后，收集L3蜕皮后2小时内的第三龄幼虫;这个程序给出0-2 hAL3E幼虫。 </li><li>转移上演幼虫填充用磷酸盐缓冲盐水（PBS）培养皿。 </li><li>用PBS冲洗幼虫从身体清除残留的食物。 </li></ol></li><li> 下幼虫的粗解剖解剖显微镜<ol><li>握住镊子幼虫的口钩。切断幼虫体在使用微剪刀身长度的前三分之一。 </li><li>保持前体幼虫的切割端与一对钳子的。使用另一对钳子的，轻轻推头朝向所述主体的内部的尖端;此过程变成幼虫体内出来。 注:此步骤可以跳过1 日龄幼虫的解剖。 </li><li>重复步骤1.2.1-1.2.2额外的幼虫5-10分钟。 注:幼虫的数量应等于或小于20，以节省时间清扫。 </li></ol></li><li> 幼虫圆角解剖 注意:此方法允许保持组织的位置保持不变。 <ol><li>仅1小时，离开水的幼虫。幼虫被固定由于窒息。 </li><li>把幼虫背侧向上在PBS中的液滴上的硅涂菜，以其背的一面朝上。 注:背侧具有纵向运行2个气管导管。 </li><li>在解剖显微镜下，插入昆虫销进入使用镊子幼虫的前尖端。拉伸幼虫身体后侧，把一个第二销到幼虫的后末端。 注意:除了昆虫针，由钨线制成的针可以是有用的21。的针可通过加热用作解剖针被嵌入在移液管尖端。 </li><li>在解剖显微镜下，使靠近与微剪刀尾一个小切口。从切口，切开背部角质层纵向沿背中线向头部。要小心，不要破坏角质层下的组织。 </li><li>伸展每一侧上的体壁角质层并将其放置4个管脚在解剖体壁的每个角上。 </li><li>用镊子除去前脂肪身体的一部分，以暴露脑-RGÇomplex在表面上露出。如有必要，除去一对唾液腺。 </li></ol></li><li> 固定并与免疫组织染色 <ol><li>把幼虫体（步骤1.2.2）的前三分之一到1.5mL微管中填充有500μL的修复溶液（4％多聚甲醛的PBS）。固定在一个时间小于20个样本，否则PBS附连到固定的样品可能会导致固定剂的不利的稀释。对于圆角解剖，除去PBS一滴，然后添加的修复方案的下降。 注:对于修复方案中，可以使用任一的3.7％甲醛或4％多聚甲醛。 </li><li>孵育在修复溶液的解剖组织，在室温下（RT）或可替代地2小时，在4℃30分钟。 </li><li>代替用500μLPBS修复溶液，并迅速冲洗样品3次。洗用500μl0.3％的PBT（PBS + 0.3％辛基苯酚乙氧基化物）样品15分钟上在r的摇臂T.对于圆角解剖，取出昆虫销和样品转移到1.5mL微管。 注意:为了提高抗体的渗透性到组织，将样品用500微升处理过的2.0％PBT 1-2小时中室温下在摇杆。 </li><li>用500μl封闭溶液（在PBT 2％牛血清白蛋白）替代PBT。孵育在室温在摇杆1.5小时的样品。 </li><li>替换用50μL的第一抗体溶液，在封闭溶液稀释即抗体封闭溶液。 </li><li>上孵育在4℃下的摇杆过夜样品。 </li><li>代替用500μL0.3％的PBT的第一抗体溶液，并迅速冲洗样品5次。用500μl0.3％的PBT，每次15分钟洗涤样品3次上在RT摇杆。 </li><li>用50μL的二级抗体溶液（在封闭溶液稀释染料缀合的抗体）替代0.3％的PBT。用于核染色，0.5μL4&#39; ，6-diamidino-2 - 苯基吲哚（DAPI，0.1毫克/毫升储液）加入到50μL的溶液。上孵育在室温或备选地在4℃下过夜2 h的摇杆的样品。 注:保持样品在黑暗中。 </li><li>代替用500μL0.3％的PBT的抗体溶液和冲洗5次。用500μl0.3％的PBT，在室温，每次15分钟洗样品3次。 </li></ol></li><li> 大脑-RG复杂的精细解剖含有PG和安装 <ol><li>使用一次性吸移管到免疫染色的样品转移到填充有0.3％PBT一个菜。 注意:为避免解剖和安装时不方便气泡，0.3％PBT可以用PBS代替。 </li><li>在解剖显微镜下，保持角质层或嘴钩与一对钳子的。使用附连到1mL注射器为"刀" 27号针，切开食管和眼盘的前尖端以去除从身体表皮大脑-RG-眼睛光盘复杂。 </li><li>国家环保总局价格从用钳子脑RG-眼睛光盘复杂食道和肠道。由于食道穿过腹神经索（VNC）以上的脑，拉出肠道后侧。 注:从样品中去除多余的成虫盘，切脑，眼光盘和腿盘之间的连接用针刀。 </li><li>重复步骤1.5.1-1.5.4其他样品。 注意:为了防止组织碎片粘到样品中，每完成样品转移到一个不同的干净盘填充有0.3％的PBT或PBS。 </li><li>转移到样品用微量干净的载玻片上的中心。 注:对于第2 次或3 龄幼虫，微量吸管尖端的端部应用刀片被截断。 </li><li>在解剖显微镜下，通过使用镊子对准他们的背侧向上个体样品。 注:RG位于脑的背侧（ 图2A）。这种对准便于在成像期间各个样本的规范。 </li><li>倾斜载玻片上并擦去之多过量PBT越好。把安装试剂一滴在载玻片上的一侧。广场下降的另一面盖玻片的边缘，并用钳子把盖玻片上的样品慢。 注:此过程防止样品从移动盖玻片外部。 </li><li>在4℃下储存样品。保持样品在黑暗中。 </li></ol></li></ol> 2.卵巢中的解剖 成年女性<ol><li> 成年女性的制备 <ol><li>饲料雌蝇与标准酵母/玉米面飞食品3-4天催肥卵巢。保持在25℃。 </li></ol></li><li> 成年女性卵巢解剖 <ol><li>麻醉雌蝇用CO 2气体并切断头。 </li><li>转移飞机构3cm的菜充满PBS。 </li><li>在解剖显微镜下，持有使用镊子背侧胸部。把握腹节A5 A6或与其他镊子和剥开朝向后侧腹部表皮。擦去从钳子的前端粘角质层。 </li><li>捏输卵管，并采取了从身体的卵巢。 </li><li>梳子和钳传播卵巢的提示。 注:此操作可以提高免疫效率。 </li></ol></li><li> 成年女性的大脑，VNC和生殖器官解剖。 注:此方法可使卵巢的神经支配可保持不变。 <ol><li>麻醉雌蝇用CO 2气体，并将它们传送到填充有PBS硅涂布培养皿中。 </li><li>在解剖显微镜下，按住长鼻和剥去头角质层钳暴露大脑。去除附着在大脑中的气管。 注:brain是白色和圆形的结构。大脑连接到VNC在胸部。 </li><li>按住背侧胸部和切断腿和翅膀一个钳子。使用其他钳子，剥去胸部腹面从腿的基地角质层。一旦VNC露出，除去附着在VNC使用镊子残留背角质层和肌肉。小心不要损伤大脑和VNC之间的连接。 </li><li>使用镊子剥去腹部表皮，露出卵巢和他们互动的器官，包括神经元，作物，肠，输卵管，子宫和附属腺体。 </li><li>除去残留的组织，包括气管和使用镊子脂肪体。 </li></ol></li><li> 固定并与免疫组织染色 <ol><li>输送约8-10对卵巢到1.5mL微管中填充有250μL的修复溶液（4％多聚在PBS ldehyde）并孵育样品在RT下30分钟。 </li><li>代替用500μLPBS修复溶液，并迅速冲洗样品3次。 </li><li>洗样品2次用500微升0.1％的PBT在RT每次5分钟。 </li><li>用50μL封闭溶液代替PBT。孵育在室温在摇杆1个小时的试样。 </li><li>替换用50μL的初级抗体溶液中的封闭溶液。 </li><li>上孵育在4℃下的摇杆过夜样品。 </li><li>代替用500μL0.1％的PBT的第一抗体溶液。用500μl0.1％的PBT，每次15分钟洗涤样品3次上在RT摇杆。 </li><li>用50μL第二抗体溶液取代0.1％PBT。对于核染色，加0.5μLDAPI至50微升的解决方案。孵育在室温在摇杆2个小时的样品。 </li><li>代替用500μL0.1％的PBT的第二抗体溶液。用500μl0.1％PBT洗涤样品3次<html>关于在室温下的摇杆，每次15分钟。 注意:洗涤过程可以在4℃进行过夜。保持样品在黑暗中。 </li></ol></li><li> 安装所述样品在载玻片上 <ol><li>转移样品到载玻片用0.1％PBT微量。 注意:为避免解剖和安装时不方便气泡，0.1％PBT可以用PBS代替。 </li><li>对于卵巢，分离在解剖显微镜下彼此用钳子卵巢管的字符串。害人之心不可含有germaria的卵巢管的提示。对于脑VNC-生殖器官复杂，除去残留的角质层，并安排在载玻片上的位置。 </li><li>擦去之多过量PBT尽可能并把安装试剂的液滴在样品的中心。将慢慢使用镊子盖玻片。 </li><li>在4℃下储存样品。保持样品在黑暗中。 </li></ol></li></ol>"&gt; 3。成像用共焦激光扫描显微镜<ol><li>观察样品，在显微镜下，并把类固醇合成的器官中的视场。当视图是固定的，所述成像系统切换到图像获取模式。 注:10-20X物镜被用于获得大脑-RG复杂或整个卵巢的图像。可替代地，40-63X物镜（水或浸油）用于可视化蛋白的亚细胞定位在GSC中或PG和卵巢的神经元神经支配。 </li><li>设置图像采集的参数上附加的软件。选择荧光的组合（ 例如 GFP和RFP）。通过快扫描过程中，调整激光功率，检测器的灵敏度（增益/偏移），和针孔的大小。 注意:为了获得在高品质的图像，扫描的激光功率和检测器（增益）的灵敏度应为每个样品进行优化。勾勒出形状的组织中，一个透射光图像可以另外被带到一个荧光图像。 </li><li>拍摄的图像的Z图库。在连续快速扫描，移动焦平面与显微镜的聚焦驱动，并设置开始位置和结束位置。切片的数量和切片之间的间隔距离被相应地调整。 </li><li>选择扫描速度，扫描方法和双向扫描模式。 </li><li> "真正开始扫描"图像采集。 </li><li>与附加软件的格式保存图像。 注:原始图像文件中包含的图像采集参数和规模的信息。导出的图像可以在计算机22上使用图像处理软件进行处理。 </li></ol>

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Syst. 89 (1), 27-34 (2014).</li><li>Yoshiyama-Yanagawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+conserved+Rieske+oxygenase+DAF-36/Neverland+is+a+novel+cholesterol-metabolizing+enzyme.">The conserved Rieske oxygenase DAF-36/Neverland is a novel cholesterol-metabolizing enzyme.</a> J. Biol. 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没有利益冲突的声明。

我们研究了蜕皮类固醇的生物合成和果蝇及其调控机制，并制定了解剖和免疫染色的协议。蜕皮甾类生物合成的定时是通过神经元输入33受环境线索，所以它是用脑，VNC，和解剖过程中其他组织沿着保持ecdysteroidogenic器官的神经支配必不可少的。 如上所述， 黑腹果蝇 PG形成与语料库allata（CA）和所述语料库cardiaca（CC）（ ?...

在多细胞生物，一组单元被赋予了在其生物活性的特殊功能是整个人体不可缺少的。为了履行自己的使命，每一个组织或器官表达了一系列有关其功能的基因，并与其他组织进行通信，以协调在发展的背景下开展活动。为了描述这种专门的细胞功能和脏器间的相互作用，我们需要与其他类型的细胞被保持完整的多架构沿着指定一组细胞。 这种专门的器官的一个例子是类固醇合成器官，许多生物合成酶介导的转化步骤从胆固醇活性类固醇激素1。大多数这些酶的基因的类固醇在器官特异性表达，并且所述生物合成途径被紧紧地通过经由体液输入和神经元的输入许多外部刺激调节。一旦合成类固醇激素分泌到血淋巴和靶向许多组织和器官，用于调节各种基因2的表达。因此，类固醇激素的作用诱导全身性反应保持动态平衡，生长和繁殖。 为了研究类固醇激素的生物合成的功能和类固醇激素的多效性的动作， 果蝇可被用作一个合适的模型系统。在幼虫期，昆虫类固醇激素，蜕皮甾类，在专门的内分泌器官生物合成称为前胸腺（PG）3。在PG，几个ecdysteroidogenic酶特异性催化多个转换步骤从胆固醇蜕皮激素，其控制在适当的发育阶段4蜕皮和变态。因此，在蜕皮甾体滴度的动态变化被调节通过响应于环境暗示许多信号传导途径。在另一方面，在成人阶段，蜕皮甾类起着生理学重要作用，包括复制，睡眠，记忆和寿命5，6，7，8。已知的是，蜕皮甾类正在积极生物合成在卵巢，调节卵子发生6，7，8，9，10，11的进展。最近，我们报道了生殖系干细胞（GSC中）的数目由蜕皮激素和性别肽信令响应于配合影响刺激12。 D.功能强大的工具果蝇遗传学和细胞生物学，包括注释良好的基因组信息，二进制基因表达系统和转基因的RNAi技术，使我们能够鉴定基因必需在PG和卵巢13，14，15到蜕皮甾类生物合成。一旦ecdysteroidogenic基因鉴定，这些基因和基因产物的动态本地化的转录调控可以在生物合成途径16进行检查。为了这个目的，定量反转录PCR，RNA 原位杂交，免疫组织学和分析中进行。这些技术的应用包括一项艰巨的任务;在PG或卵巢的精细解剖。特别地，果蝇的PG比其他昆虫的相对较小（ 例如 ，蚕和吹飞），因此人们需要能够实践水果的至关重要的技能飞清扫采样。此外，无论是ecdysteroidogenic机关收到支配期从中枢神经系统（CNS）17，18，19，20。因此，准确的解剖分析，在ecdysteroidogenic机关应保持与中枢神经系统和其他器官，不破坏他们的神经连接沿完好无损。 在这里，我们提供的解剖和D中类固醇合成器官的可视化协议。 果蝇。学习解剖技术是这些实验的关键起点。此外，可以成功地标记类固醇合成器官以及它们之间的互动与器官的几种抗体和GAL4驱动程序行。以这些技术，材料和遗传学的优势，可以研究类固醇激素合成的综合机制。

<table><tbody><tr><td>&lt;strong&gt;egg collection&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>tissue culture dish (55 mm)</td><td>AS ONE</td><td>1-8549-02</td><td>&amp;nbsp;for grape-juice agar plates</td></tr><tr><td>collection cup</td><td>HIKARI KAGAKU</td><td></td><td></td></tr><tr><td>yeast paste</td><td>Oriental dry yeast, Tokyo</td><td></td><td></td></tr><tr><td>100% grape juice</td><td>Welch Food Inc.</td><td></td><td></td></tr><tr><td>&lt;strong&gt;rearing larvae&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>small vials (12ml, 40&amp;times;23.5 mm, PS)</td><td>SARSTEDT</td><td>58.487</td><td></td></tr><tr><td>disposable loop</td><td>AS ONE</td><td>6-488-01</td><td></td></tr><tr><td>standard fly food</td><td></td><td></td><td>&amp;nbsp;the recepi us on the website of Blooington stock center.</td></tr><tr><td>&lt;strong&gt;dissection&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>dissecting microscope</td><td>Carl Zeiss</td><td>Stemi 2000-C</td><td></td></tr><tr><td>dissecting microscope</td><td>Leica</td><td>S8 AP0</td><td></td></tr><tr><td>tissue culture dish (35 x 10 mm, non-treated)</td><td>IWAKI</td><td>1000-035</td><td></td></tr><tr><td>Sylgard</td><td>TORAY</td><td></td><td>coarting silicon inside dishes</td></tr><tr><td>Terumo needle (27G, 0.40 x 19 mm)&amp;nbsp;</td><td>TERUMO</td><td>NN-2719S</td><td>A &quot;knife&quot; to cut the tissue</td></tr><tr><td>forceps, Inox, #5</td><td>Dumont, Switzerland</td><td></td><td></td></tr><tr><td>insect pin (0.18 mm in diameter)</td><td>Shiga Brand</td><td></td><td>for fillet dissection</td></tr><tr><td>micro scissors</td><td>NATSUME SEISAKUSHO CO LTD.&amp;nbsp;</td><td>MB-50-10</td><td></td></tr><tr><td>&lt;strong&gt;fixation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>ultrapure water</td><td>Merck Millipore</td><td></td><td></td></tr><tr><td>phosphate buffered saline (PBS)</td><td></td><td></td><td></td></tr><tr><td>Formaldehyde</td><td>Nacalai tesque</td><td>16222-65</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Nacalai tesque</td><td>02890-45</td><td></td></tr><tr><td>Triton-X100</td><td>Nacalai tesque</td><td>35501-15</td><td></td></tr><tr><td>microtubes (1.5 ml)</td><td>INA OPTIKA</td><td>CF-0150</td><td></td></tr><tr><td>&lt;strong&gt;Incubation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>As one swist mixer TM-300 (rocker)</td><td>As one</td><td>TM-300</td><td>rocker</td></tr><tr><td>Bovine Serum Albumin</td><td>SIGMA</td><td>9048-46-8</td><td></td></tr><tr><td>&lt;strong&gt;primary antibody&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-Sro (guinea pig), 1:1000</td><td></td><td></td><td></td></tr><tr><td>anti-GFP (rabbit), 1:1000</td><td>Molecular Probes</td><td>A6455</td><td>Shimada-Niwa ans Niwa, 2014</td></tr><tr><td>anti-GFP (mouse mAb, GF200), 1:100</td><td>Nakarai tesque</td><td>04363-66</td><td></td></tr><tr><td>anti-5HT (rabbit), 1:500</td><td>SIGMA</td><td>S5545</td><td></td></tr><tr><td>anti-Hts 1B1 (mouse)</td><td>Developmental Studies Hybridoma Bank (DSHB)</td><td>1B1</td><td></td></tr><tr><td>anti-DE-cadherin (rat), 1:20</td><td>DSHB</td><td>DCAD2</td><td></td></tr><tr><td>anti-nc82 (mouse), 1:50</td><td>DSHB</td><td>nc82</td><td></td></tr><tr><td>&lt;strong&gt;secondary antibody&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td><td>Life Technologies</td><td>A-11008</td><td></td></tr><tr><td>Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate</td><td>Life Technologies</td><td>A-11001</td><td></td></tr><tr><td>Goat anti-Rat IgG (H+L) Secondary Antibody, Alexa Fluor 546 conjugate</td><td>Life Technologies</td><td>A-11081</td><td></td></tr><tr><td>Goat anti-Guinea Pig IgG (H+L) Secondary Antibody, Alexa Fluor 555 conjugate</td><td>Life Technologies</td><td>A-21435</td><td></td></tr><tr><td>Alexa Fluor 546 dye-conjugated phalloidin</td><td>Life Technologies</td><td>A-22283</td><td></td></tr><tr><td>&lt;strong&gt;Mounting reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Micro slide glass</td><td>Matsunami Glass Ind.,Ltd.</td><td>SS7213</td><td></td></tr><tr><td>Square microscope cover glass</td><td>Matsunami Glass Ind.,Ltd.</td><td>C218181</td><td></td></tr><tr><td>FluorSave reagent (Mounting reagent)</td><td>Calbiochem</td><td>345789</td><td></td></tr><tr><td>Transfer pipette 1 ml (Disposable dropper)</td><td>WATSON</td><td>5660-222-1S</td><td></td></tr><tr><td>&lt;strong&gt;imaging&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>LSM700 laser scanning microscope system</td><td>Carl Zeiss</td><td></td><td>inverted Axio Observer. Z1 SP left</td></tr><tr><td>&lt;strong&gt;image processing&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>LSM700 ZEN</td><td>Carl Zeiss</td><td></td><td>It is a special user interface based on the 64 bit Microsoft Windows7 operating system</td></tr><tr><td>ImageJ</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Fly stocks&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>w; GMR45C06-GAL4</td><td></td><td></td><td>&amp;nbsp;from Bloomington Drosophila Stock Center. (#46260)</td></tr><tr><td>UAS&amp;ndash;GFP; UAS&amp;ndash;mCD8::GFP</td><td></td><td></td><td>gifts from K. Ito, The University of Tokyo.</td></tr><tr><td>w[1118]</td><td></td><td></td><td></td></tr><tr><td>w; phantom-GAL4#22/UAS-turboRFP</td><td></td><td></td><td></td></tr><tr><td>w; UAS-mCD8::GFP; TRH-GAL4</td><td></td><td></td><td>see in Ref29, Alekseyenko, O. V, Lee, C. &amp;amp; Kravitz, E. A.(2010)</td></tr><tr><td>w; UAS-mCD8::GFP</td><td></td><td></td><td>&amp;nbsp;from Bloomington Drosophila Stock Center. (#32188)</td></tr><tr><td>yw;; nSyb-GAL4</td><td></td><td></td><td>&amp;nbsp;from Bloomington Drosophila Stock Center. (#51941)</td></tr></tbody></table>

protocols for visualizing steroidogenic organs and their interactive organs with immunostaining in the fruit fly drosophila melanogaster

在多细胞生物体中，一小群细胞被赋予了在其生物活性的专门功能，诱导生长繁殖全身反应。在昆虫幼虫前胸腺（PG）和生物合成的主要类固醇激素称为蜕皮类的成年女性卵巢发挥重要作用。这些ecdysteroidogenic器官来自神经系统，通过它合成的时间是由环境因素影响的支配。在这里，我们描述了可视化ecdysteroidogenic机关和幼虫它们之间的互动器官和果实蝇成虫果蝇 ，这为研究类固醇激素的生物合成及其调控机制的合适模型系统的协议。熟练的解剖，我们保持着ecdysteroidogenic器官的位置和它们互动的器官，包括大脑，腹神经索和其他组织。用免疫染色针对ecdysteroidogenic酶ntibodies，通过组织特异性启动子的转基因驱动的荧光蛋白质一起，是可用来标记ecdysteroidogenic细胞。此外，ecdysteroidogenic器官的支配，也可以通过特异性抗体或多种类型的神经元GAL4驱动程序的集合标记。因此，ecdysteroidogenic机关及其神经元连接可以同时通过免疫组化和转基因技术，可视化。最后，我们将介绍如何可视生殖干细胞，其增殖和维护由蜕皮类控制。这种方法有助于类固醇激素的合成及其神经调节机制的全面了解。

我们使用了上述协议，以可视化类固醇合成器官和果蝇幼虫和成年女性的互动器官。协议的总体方案示于图1。 的RG，包括PG（ 图2D），是比大脑更小和更透明的，并且位于大脑（ 图2A-C和3A-E）的前侧-背侧。以标记PG细胞，几组都产生各种类型的针对ecdysteroidogenic酶的抗体（ 即 ，梦幻岛23，Spookier 24，...

Watch this Scientific Journal Video about Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster at JoVE.com

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

我们描述了解剖，固定，并在果蝇幼虫和成年女性类固醇合成器官，研究类固醇激素的生物合成及其调控机制的免疫染色的协议。除了类固醇合成器官，我们可以形象地类固醇合成器官的神经支配和类固醇合成的靶细胞，如生殖系干细胞。

协议的可视化类固醇机关及其互动器官在果蝇免疫染色<em&gt;果蝇</em

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

protocols-for-visualizing-steroidogenic-organs-their-interactive

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

10.4K 次观看.  University of Tsukuba. 我们描述了解剖，固定，并在果蝇幼虫和成年女性类固醇合成器官，研究类固醇激素的生物合成及其调控机制的免疫染色的协议。除了类固醇合成器官，我们可以形象地类固醇合成器官的神经支配和类固醇合成的靶细胞，如生殖系干细胞。 

视频: Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

科研

发育生物学

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

果蝇蛹卵巢的解剖与染色

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

荧光标记的果蝇心结构

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

生物学

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

早期果蝇胚胎内免疫组织化学和RNA原位分布的方案

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...

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 ...

Imaging Centrosomes in Fly Testes

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell differentiation. Centrosomes are essential to determine the cell division axis during mitosis and to nucleate cilia during interphase. The identity of the proteins that mediate these dynamic changes remains only partially known, and the function of many of the proteins that have been implicated in these processes is still rudimentary. Recent work has shown that Drosophila spermatogenesis provides a powerful system to identify new proteins critical for centrosome function and formation as well as to gain insight into the particular function of known players in centrosome-related processes. Drosophila is an established genetic model organism where mutants in centrosomal genes can be readily obtained and easily analyzed. Furthermore, recent advances in the sensitivity and resolution of light microscopy and the development of robust genetically tagged centrosomal markers have transformed the ability to use Drosophila testes as a simple and accessible model system to study centrosomes. This paper describes the use of genetically-tagged centrosomal markers to perform genetic screens for new centrosomal mutants and to gain insight into the specific function of newly identified genes.

Imaging of centrosomal proteins during Drosophila spermatogenesis is a powerful method to identify new proteins critical for centrosome biology as well as to elucidate the particular function of known players in this process.

在飞睾丸成像中心体

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell ...

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

Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and organogenesis. Immunostaining allows for the visualization of developing tissues and organs. However, a protective cuticle that forms at the end of embryogenesis prevents permeation of antibodies into late-stage embryos and larvae. While dissection prior to immunostaining is regularly used to analyze Drosophila larval tissues, it proves inefficient for some analyses because small tissues may be difficult to locate and isolate. Sonication provides an alternative to dissection in larval Drosophila immunostaining protocols. It allows for quick, simultaneous processing of large numbers of late-stage embryos and larvae and maintains in situ morphology. After fixation in formaldehyde, a sample is sonicated. Sample is then subjected to immunostaining with antigen-specific primary antibodies and fluorescently labeled secondary antibodies to visualize target cell types and specific proteins via fluorescence microscopy. During the process of sonication, proper placement of a sonicating probe above the sample, as well as the duration and intensity of sonication, is critical. Additonal minor modifications to standard immunostaining protocols may be required for high quality stains. For antibodies with low signal to noise ratio, longer incubation times are typically necessary. As a proof of concept for this sonication-facilitated protocol, we show immunostains of three tissue types (testes, ovaries, and neural tissues) at a range of developmental stages.

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 ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

解剖和免疫染色成虫盘的<em&gt;果蝇</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm development. However, immunolabeling of the whole Drosophila testis is often associated with significant non-uniformity of staining due to antibody penetration. Squashed preparations only partially overcome the problem since it decreases the 3D quality of the analyses. Herein, we describe a whole-mount protocol using NP40 and heptane during fixation together with immunolabeling in liquid media. It preserves the volume suitable for confocal microscopy together with reproducible and reliable labeling. We show different examples of 3D reconstitution of spermatocyte nuclei from confocal sections. The intra- and inter-testes reproducibility allows 3D quantification and comparison of fluorescence between single cells from different genotypes. We used different components of the intranuclear MINT structure (Mad1-containing Intra Nuclear Territory) as well as two components associated with the nuclear pore complex to illustrate this protocol and its applications on the largest cells of the testis, the S4-S5 spermatocytes.

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

We present here an improved protocol for the whole-mount preparation and immunostaining of Drosophila testes, suitable for confocal microscopy and also allowing reproducible and reliable labeling. We illustrate this protocol by 3D representation and quantification of colocalization experiments on the large S4-S5 spermatocytes.

用于大型精子细胞三维共焦分析的全山 嗜血杆菌 睾丸免疫染色

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm ...

Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands

Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila melanogaster. Therefore, it is an excellent model to study heterochromatin formation and maintenance. Polytenized cells, such as the ones found in salivary glands of third instar D. melanogaster larvae, provide an excellent tool to observe the chromatin amplified nearly a thousand times and have allowed researchers to study changes in the distribution of heterochromatin in the nucleus. Although the observation of heterochromatin components can be carried out directly in polytene chromosome preparations, the localization of some proteins can be altered by the severity of the treatment. Therefore, the direct visualization of heterochromatin in cells complements this type of study. In this protocol, we describe the immunostaining techniques used for this tissue, the use of secondary fluorescent antibodies, and confocal microscopy to observe these heterochromatin aggregates with greater precision and detail.

Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands

This protocol aims to visualize heterochromatin aggregates in Drosophila polytene cells.

免疫荧光染色，用于 在果蝇 唾液腺中可视化异色素相关蛋白质

Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila ...

协议的可视化类固醇机关及其互动器官在果蝇免疫染色

摘要

探索更多视频

此视频中的章节

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

果蝇蛹卵巢的解剖与染色

荧光标记的果蝇心结构

早期果蝇胚胎内免疫组织化学和RNA原位分布的方案

夹层和染色果蝇幼虫卵巢

在飞睾丸成像中心体

后期阶段的胚胎和幼虫的超声，促进免疫荧光染色

解剖和免疫染色成虫盘的

用于大型精子细胞三维共焦分析的全山嗜血杆菌睾丸免疫染色

免疫荧光染色，用于在果蝇唾液腺中可视化异色素相关蛋白质

协议的可视化类固醇机关及其互动器官在果蝇免疫染色

摘要

探索更多视频

此视频中的章节

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

果蝇蛹卵巢的解剖与染色

荧光标记的果蝇心结构

早期果蝇胚胎内免疫组织化学和RNA原位分布的方案

夹层和染色果蝇幼虫卵巢

在飞睾丸成像中心体

后期阶段的胚胎和幼虫的超声，促进免疫荧光染色

解剖和免疫染色成虫盘的

用于大型精子细胞三维共焦分析的全山 嗜血杆菌 睾丸免疫染色

免疫荧光染色，用于 在果蝇 唾液腺中可视化异色素相关蛋白质

用于大型精子细胞三维共焦分析的全山嗜血杆菌睾丸免疫染色

免疫荧光染色，用于在果蝇唾液腺中可视化异色素相关蛋白质