我想感谢詹妮弗柯蒂斯博士在图图片。 1，批判性阅读的手稿杰里米费根博士和佛罗伦萨马洛。我们的工作是支持由NIH资助1R01GM088202。

1。飞头夹层<ol><li>确保你手头上的所有材料（包括明胶涂层幻灯片）。准备戊二醛和锇固定液（见下文）。每基因型嵌入，分装在1.5毫升冰管200μL戊二醛修复方案。 </li><li>麻醉的CO 2垫（一个头是在过程中被摧毁，我们通常解剖七个飞头嵌入的情况下，每基因型6）上的苍蝇。 </li><li>保持和稍微按胸部飞，背侧，镊子，用锋利的手术刀切断的头部。 </li><li>稳定飞头颈部接触镊子，小心地切了一只眼的一小部分。这增强了固定液的渗透（如果你打算使用克隆分析你的眼睛部分，削减与没有或较小的克隆的眼睛）。避免触摸和破坏完整的眼睛，稍后将切片。 </li><li>触摸切断眼表面的镊子或手术刀头和转移冰头戊二醛/磷酸固定液（元首经常漂浮在固定液上）解剖的元首不应该保持更长的时间修复在戊二醛超过15分钟前锇此外。与同一基因型的其他苍蝇重复步骤1.2 - 1.5。 </li></ol> 2。固定和嵌入（所有步骤中使用的手套！） <ol><li>自旋为1分钟，以10,000 rpm的Eppendorf离心机flyheads。继续即使头不下沉。 </li><li> OSO 4溶液中加入200μL和修复至少30分钟，长达1小时在冰上。 </li><li>使用巴斯德吸管，去除戊二醛/锇的解决方案（请务必使用适当的废物处理程序！）和200μL锇溶液取代。在冰上孵育1-6小时。始终确保磁头与液体完全覆盖，永远不会删除所有的解决方案作为元首或眼睛可能崩溃！ </li><li>使用巴斯德吸管，弃固定液，在冰上至少5分钟增加0.5 -1毫升30％的乙醇和孵化（确保使用适当的废物处理程序，因为现在的乙醇包含锇！）。 </li><li> 50％，70％（80％），90％和100％的两倍乙醇重复上述步骤。如果用电子显微镜分析的眼睛，包括80％的乙醇一步。 70％的清洗步骤后，样品可以去除冰。 </li><li>更换与环氧丙烷（易挥发，继续努力，在引擎盖）等体积的乙醇​​。孵育10分钟，在室温。 </li><li>重复环氧丙烷洗。分批工作，如果并行处理的许多基因型，以避免崩溃的眼睛，由于环氧丙烷蒸发。 </li><li>删除最环氧丙烷和使用塑料移液管，增加约500μL1:1的树脂:环氧丙烷的解决方案。在室温下，平衡过夜。 </li></ol> 3。在模具的嵌入和安排<ol><li>确保所有模具都贴上标签，保持跟踪您的不同基因型，然后填写100％的树脂模具。不要溢出（没有"高山"直整个模具表面时，在模具表面）。使用硬树脂EM分析。 </li><li>使用移液管，除去50％的树脂元首，取代100％的树脂，并在室温下孵育3-4小时。主管与树脂渗透，他们将沉到试管底部。 </li><li>使用牙签已经打在坚硬的表面，一旦创建一个小钩的，在时间转移到一个模具一个头。 </li><li>铝箔放在工作区的解剖范围，以防止树脂泄漏。 </li><li>用解剖针，虽然你的范围，漩涡在树脂的头微微仔细将其移动到模具底部接近尖端。 </li><li>小心地将完整的（！）的眼睛（或你的未来如果你想有截面切片平面）颈部表面与模具的前墙，一定要保持眼睛的切线表面大约半个脑袋离模具壁的直径。在极少数情况下，眼球表面和头部的角质层之间的空白处，可看到一只眼睛崩溃。如果你有一个折叠的眼睛，取代它的第 7 次备用眼。 </li><li>重复所有首长。一旦完成所有的路线，重新检查所有的目光，以确保他们不动，当您移除树脂针。 </li><li>如果你有麻烦与眼睛移动，当您删除解剖针，位置所需的眼睛，迅速收回针稍稍远离头部，然后慢慢松开针完全。 </li><li>烘烤模具一夜之间在70 ° C。 </li></ol> 4。修整和切片<ol><li>移除弯曲模具淬硬模具块，Keeping跟踪哪个块对应的基因型（如存储在小闪烁瓶）。 </li><li>修剪，使用适合您的切片机，是装在一个树脂玻璃或铝块面对夹头。山块修剪，夹头的眼睛，。 </li><li>在使用聚四氟乙烯涂层剃须刀的清扫范围的吩咐，小心地切断你的格挡只有顶层。这会留下一个清晰的表面，虽然你应该看到的眼睛，因此可以预测未来切片平面。避免削减你的手指！ </li><li>切断头部两侧和前面多余的塑料（即用于模具的顶部，对准眼睛时），直到头部周围的塑料大部分被切断。最好是慢慢接近头部，切断多个塑料薄层。 </li><li>使用干净的刀片，从眼睛上方小心取出你要节后面（通常切圆的眼睛，在一个很小的角度，原来的模具表面）在精确的平面超薄层。 </li><li>继续，直到你刚开始切去眼球的外表面。确保您使用刀片的清洁区，以获得光泽，透明的表面，这使得在切片更容易对齐。 </li><li>为了节省刀片，各地块的两侧修剪原油的刀片和老年人使用的精细剪裁（先切和4.6）的一个新的刀片。最后，您将有一个三面略有倾斜，切断顶部对应到你的未来截面金字塔。 </li><li>安装在切片块。实际切片将切片类型而不同，并不会在这里详细介绍。我们用一个Sorvall MT5000一个HISTO质量钻石刀，切0.5〜1μm的部分。光镜下分析，截面厚度略有差异并不重要。如果切片白+基因，第一个克隆标记1μm的部分，以确保有足够的色素颗粒相邻（图2）每节rhabdomeres。 </li><li>路段将浮在水面上。提起了一个木制的棉条从下面的水在旋转运动表面的扁平结束前20-30节，和他们转移到一滴水明胶镀膜玻璃幻灯片（上加热板保持在100℃左右）。 </li><li>重复20-30节。等待，直到水蒸发和部分粘到幻灯片。通常情况下，3只眼的部分安排在三列双程证行（三只眼）（顶部/底部），放在一张幻灯片。 </li></ol> 5。染色和显微镜分析<ol><li>除非，切片克隆，染色部分。将滑动部分设置在加热块在90 ° C。甲苯胺蓝染色解决方案免除到0.22μm的过滤器连接到幻灯片从30毫升注射器和染色10秒。立即用蒸馏水冲洗广泛。空气干燥。 </li><li>用塑料移液管，分发3滴的DPX安装介质上的幻灯片，并盖上盖玻片。顶部与3助攻，让安装介质（例如在室温过夜）硬化。 </li><li>光学显微镜的图像。使用5倍或10倍镜头找到一个很好的部分，然后进行详细的分析和拍照切换到63X油浸镜头。我们通常使用相衬的设置，但暗场光学系统的工作，以及。 </li></ol> 6。代表性的成果: 最常见的，眼睛是内嵌在一个塑料作为一种遗传屏幕的一部分或在测试与已知基因的影响无论是一般的眼睛结构或极性遗传相互作用的过程中检测到的外部粗糙眼表型的详细分析。眼睛部分的典型结果如图3所示。野生型的小眼（图3A）显示完整的感光补充色素细胞晶格包围。相反，在一个斜视 （AKA客梵高7 stbm，）突变体，ommatidial极性丢失，即使感光补充目前，旋转和手性是随机的（图3B）。此外，自stbm等位基因切片在AW -背景，色素颗粒在图失踪。 3B。图3C显示克隆分析的一个例子。果蝇Rho激酶（drok）ommatidial旋转以及 8眼结构的完整性的要求。色素的色素细胞和rhabdomeres（通常的情况下可以通过确定纯 合子 drok 2克隆， 克隆使用无眼，工党和一个P -元素补充的背景下瓦特强烈表达的白色标记基因-在野外突变诱导型和杂合组织）。因此，这是可能的，以确定他们缺乏色素突变的地区。由于色素沉着的细胞自主的R - CELLS提到，个别的R -细胞的基因型可确定（见箭头在图3c）。 <img src="/files/ftp_upload/2959/2959fig1.jpg" alt="图1"/> 图1。 果蝇的眼睛是极好的模型系统，因为生物学家，相反的野生型眼（一）表面光滑，突变的眼表面的外部经常粗糙（二），表明潜在的眼表型。在所有的数字，前左和背。图片礼貌，美国，新墨西哥州拉斯克鲁塞斯，新墨西哥州立大学博士詹妮弗柯蒂斯。 <img src="/files/ftp_upload/2959/2959fig2.jpg" alt="图2"/> 图2单小眼切片的光镜图像，使用所描述的方法。 R7的R8之上的谎言，因为只有七个R -细胞在每ommatidium时可见。 （A"）在R7，R7的细胞体内检测到R1和R6（黄色箭头"）之间。相比之下，在R8的水平（二），R8的细胞体是R1和R2之间（黄色箭头在B&#39;）检测。蓝箭头标记可以用来作为一个自治细胞色素标记的色素颗粒。 <img src="/files/ftp_upload/2959/2959fig3.jpg" alt="图3"/> 图3。通过野生型（A）stbm（B）和drok（C）突变的成年果蝇眼睛的切线部分。下面的部分原理图显示极性的小眼（见箭头（一））。圆圈代表小眼感光补充的缺陷。黄线代表的背/腹对称线（赤道）。相反的野生型（一）面向小眼，平面组织失去了在stbm突变（二）。注意stbm突变节中缺乏色素由于其W -突变的背景。 （三）由于drok基因突变是致命的，他们必须在克隆分析。因此，色素细胞是野生型或杂合子（填充蓝色箭头），而缺乏的R -细胞色素（打开蓝色箭头）是纯合子突变。除了 ​​旋转缺陷，drok突变体也表现出结构性缺陷，包括R -细胞的缺失或多余的号码。请注意，克隆分析，部分不染，以避免模糊的色素颗粒。

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没有利益冲突的声明。

用果蝇作为模式生物，导致遗传筛选确定的包括人类在内的高等真核生物中高度保守的信号途径所必需的基因家族的创始成员的许多。一直以来，在​​实验室条件下，功能的眼睛是为生存而可有可无，映入眼帘的是一个新基因功能的发现和基因网络的评估特别适合的组织。超微结构分析的飞眼睛使用的描述方法，从而导致发育和疾病相关的基本发现。最初，单细胞克隆进行分析，利用X射线诱导密切相关的隐性标记的细胞自治相结合，克隆项目。最近，产生克隆的FLP /首次登记税制度的可用性大大促进眼睛部分 6,9致死突变的表型分析。 眼睛部分的分析并不局限于这里所描述的切线节。如果需要的话，磁头可以在任何方向对准，在模具和横截面可研究在头层和髓质等深层。因此，这里所描述的协议是一个通用的方法对眼睛和头部结构果蝇成人的分析。

通用设备: <ul><li>对于一般飞牧， 看到 10 </li><li>标准解剖镜下（如尼康SMZ - 645立体声变焦显微镜），最好有一个环形光源，以防止铸造你的手的影子。 </li><li> CO 2的飞板，3MM的Whatman纸覆盖在清扫，防止灰尘提供保护。 </li><li>明胶涂层幻灯片:10G明胶和1G CrKSO 4 × 12 H 2 O溶解在加热板的1L水（烧开，几乎带来大约需要2小时） 。持有成明胶溶液，并用铝箔纸覆盖过夜风干浸以及洗（肥皂）和冲洗（DH 2 O）的幻灯片。 </li><li>加热炉（70 ° C;旧真空过滤干燥硝化棉使用微波炉以及适合这个目的）。 </li><li>切片机超薄切片（0.5-1微米厚）的能力，如电子显微镜设施使用。我们用一个Sorvall MT5000 Ultramicrotome一个HISTO质量钻石刀。玻璃刀也能工作。 </li></ul> 固定的解决方案: <ul><li>准备一个0.2 M磷酸钠缓冲液，pH值7.2的原液。 </li><li>准备在0.1M磷酸钠缓冲液，pH值7.2（可存放在4℃的4个星期）2％戊二醛。至少要嵌入每基因型200μL。置于冰上。 </li><li>锇的解决方案:4 OSO在0.1M磷酸钠缓冲液，pH值7.2 2％。只够要嵌入每基因型200μL，因为该解决方案无法储存。置于冰上。 </li></ul>戊二醛和OSO 4具有很强的毒性，应在通风良好的引擎盖格外小心处理。使用过滤嘴，在处理 OSO 4时，以防止您的吸管发黑。 脱水:设为30％，50％，70％（80％），90％和100％的乙醇溶液。最好凉爽的30％，50％，70％在使用前冰乙醇。 染色液: 1％甲苯胺蓝在1％的硼砂。 <table border="1"><tbody><tr><th></th><th>软</th><th>硬盘</th></tr><tr><td>树脂A </td><td> 54克</td><td> 50克</td></tr><tr><td>固化剂乙</td><td> 44.5克</td><td> 50克</td></tr><tr><td>加速器彗星</td><td> 2.5克</td><td> 1.75克</td></tr><tr><td>增塑剂ð </td><td> 10克</td><td> 0.75克</td></tr></tbody></table> 表1。树脂的制备 为了准备树脂，仔细，但使用大型搅拌棒磁力搅拌器彻底混合在一个塑料烧杯中的所有成分。避免气泡。混合后，分装于-20 ° C标准的闪烁瓶或类似容器，并冻结使用软树脂为所有的应用程序，除非您的EM工厂要求更难制定。用手套处理的树脂（其unpolymerized形式致癌）。聚合设备和废物的树脂，烘烤在70 ° C过夜。可能会被安全地丢弃废物，设备清洗和重用。溅出unpolymerized树脂可以用异丙醇清洗。 <table border="1"><tbody><tr><th>试剂</th><th>供应商</th><th>目录编号</th></tr><tr><td>飞垫</td><td>例如杰纳西</td><td> 59-119 </td></tr><tr><td>戊二醛</td><td>西格玛</td><td> G7526 - 10 × 10毫升</td></tr><tr><td> OSO 4 </td><td> Polysciences，公司</td><td> 0972A - 20 </td></tr><tr><td> Propyleneoxide </td><td>费舍尔</td><td> 04332-1 </td></tr><tr><td>手术刀处理，＃3 </td><td>费舍尔</td><td> 22080046 </td></tr><tr><td> ＃11手术刀片</td><td>费舍尔</td><td> 08 - 916 - 5B </td></tr><tr><td>转让移液器</td><td>费舍尔</td><td> 13 - 711 - 7M </td></tr><tr><td> Durcupan（R）的ACM树脂</td><td>西格玛（Fluka公司） </td><td> 44610 - 1EA </td></tr><tr><td>钢解剖针</td><td>费舍尔</td><td> S17346 </td></tr><tr><td> BEEM平面嵌入模具</td><td>电子显微镜科学。 </td><td> 70904-12 </td></tr><tr><td> Teflon涂层刀片</td><td>电子显微镜科学。 </td><td> 71970 </td></tr><tr><td>棉条（无菌棉签） </td><td>费舍尔</td><td> 14-959-81 </td></tr><tr><td>载玻片</td><td>费舍尔</td><td> 12-550-143 </td></tr><tr><td>盖玻片1，22X60毫米</td><td>费舍尔</td><td> 12 - 531K </td></tr><tr><td>明胶</td><td>费舍尔</td><td> ICN96010280 </td></tr><tr><td>铬（Ⅲ）K SO 4十二水合</td><td>西格玛</td><td> 243361 </td></tr><tr><td>钻石HISTO刀，6毫米</td><td> Diatome美国</td><td> 60他</td></tr><tr><td>甲苯胺蓝Ø </td><td>费舍尔</td><td> BP107 - 10 </td></tr><tr><td>硼砂（钠，四硼酸） </td><td>费舍尔</td><td> AC20629 - 1000 </td></tr><tr><td> DPX安装培养​​基</td><td>西格玛</td><td> 44581 - 100ML </td></tr></tbody></table> 表2。材料

preparation of adult drosophila eyes for thin sectioning and microscopic analysis

果蝇一直被用来作为模型系统，研究发展，主要是由于与它的基因听话的难易程度。多年来，众多的突变株和技术技巧已经制定，让复杂的问题要问，并在合理的时间内回答。到所有已知的主要信号转导通路的组成部分的相互作用的基本观点已得到正向和反向基因果蝇的研究。苍蝇的眼睛已被证明是非常好，适合突变分析，因为，在实验室条件下，苍蝇可以生存无功能的眼睛。此外，虫眼的表面是由一些个别单位800眼（面或小眼），形成了定期，表面光滑时，看着在解剖显微镜下。因此，很容易看到是否突变可能会影响眼睛发育或外部表面光滑的损失（"粗糙眼"的表型;图1）。增长或整体眼睛的大小，分别为（基于屏幕的例子外眼形态， 例如 :1） 。随后的详细分析，眼表型需要固定，塑料包埋薄切片和成人的眼睛。 果蝇眼睛的发展，从所谓的眼成虫光盘，一包上皮细胞增殖，并在幼虫和蛹的阶段区分（审查， 如 2） 。每个ommatidium由20个细胞，其中包括8个光感受器（PR或R -细胞，图2），4个镜头分泌的视锥细胞，色素细胞（"六角"周围的R -细胞集群）和猪鬃。每个ommatidium的光感受器，最容易确定其对光敏感的细胞器，rhabdomeres，是一个梯形六"外"（R1 - 6）和两个"内在的"光感受器（R7 / 8举办R8 [图2]下面是R7的，因此只在更深的领域从眼球的部分）。每个面的梯形是其邻国和整体正位眼（图3A）和背腹轴的精确对准。特别是，背，腹（黑色和红色箭头，分别）半眼的小眼是互为镜像，对应在平面细胞极性的信号（ 审查见如3）建立了两个手性形式。 这里描述的方法来生成半薄眼的部分（如在图3中提出的）从最初Tomlinson和准备 4描述的略有修改。它允许所有细胞的形态分析，除了透明的视锥细胞。此外，色素的R -细胞（蓝色箭头在图2和3）可以用来作为一个R -细胞的基因型的细胞自主的标志，在一个子集的R -细胞可以遗传的基因因此要求很容易地确定5,6。

Watch this Scientific Journal Video about Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis at JoVE.com

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

准备成人的标准方法<em&gt;果蝇</em半薄切片，光镜下分析&gt;的眼睛是在这里。该协议可用于眼部缺陷的形态分析总值，或可以用在眼睛特定的细胞类型（如光感受器的克隆分析）或电子显微镜分析，以确定基因的遗传要求与指示调整。

成人的制备果蝇</em薄切片和显微镜分析>的眼睛

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

preparation-adult-drosophila-eyes-for-thin-sectioning-microscopic

Universidad San Sebastian

Research

JoVE Journal

Biology

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

16.1K 次观看.  Albert Einstein College of Medicine. 准备成人的标准方法果蝇的眼睛是在这里。该协议可用于眼部缺陷的形态分析总值，或可以用在眼睛特定的细胞类型（如光感受器的克隆分析）或电子显微镜分析，以确定基因的遗传要求与指示调整。

视频: Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI样品制备：超薄层的方法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

科研

生物学

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue.  Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle.  Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm.  We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections.  The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed.  We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice.  This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice.  A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT).  This is then quickly frozen with crushed dry ice and the resulting  wafer  can be position easily for cryostat sectioning.  Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

薄切片进行免疫组化切片的筹备工作

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

Whole Cell Recordings from Brain of Adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown.  The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.

This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording.

从整个成年果蝇大脑细胞记录

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

高分辨率视频跟踪移动的，在成年果蝇。

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

从成年小鼠海马齿状回的剖析

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

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 ...

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 ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

发展和成人的制备果蝇大脑和视网膜实时成像

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Preparation of Drosophila S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells.
S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24&deg;C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks.
Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.

Preparation of Drosophila S2 cells for Light Microscopy

Drosophila Schneider (S2) cells are an increasingly popular system for the discovery and functional analysis of genes. Our goal is to describe some of the microscopic techniques that make S2 cells such an increasingly important experimental system.

制备果蝇 S2细胞，光学显微镜

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

成人的制备果蝇的眼睛

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