内部机构的资金和在密歇根大学的生物科学学者计划支持这项工作。

蠕虫病毒的制备<ol><li> L4幼虫蠕虫传输到适当板18 - 24小时前成像22。这将确保你有鸡蛋供应充足，为早期胚胎轴承成人。 </li><li>准备凡士林和琼脂糖管。我们准备在一个烧杯中的微波熔化的凡士林，并配发到每管4-5毫升等分几个15ML猎鹰管。同样融化2-3％（W / V）琼脂糖缓冲区（M9或鸡蛋缓冲液），等分4-5毫升到猎鹰管。要小心不要煮沸过剩，以最大限度地减少蒸发。成像当日，融化在65-70℃加热块1管凡士林和1琼脂糖管在微波炉中融化，再小心，尽量减少沸腾了。商店在加热块管，以保持在熔融状态下凡士林和琼脂糖。 </li><li>设为琼脂糖垫。位置2指南幻灯片之间的新的显微镜幻灯片，这是由单一的普通实验室磁带坚持每个指南幻灯片的顶部和底部的一层覆盖的幻灯片。使用巴斯德吸管折断放在新的幻灯片2-3滴熔化琼脂糖结束。盖与第二幻灯片垂直于原始幻灯片，所以，它涵盖并有助于分散琼脂糖创建一个薄的方形垫。 第二届幻灯片休息以上的磁带上的指导幻灯片。琼脂糖的浓度可能会增加至5％，加厚垫。 </li><li>使用解剖范围，铂丝“挑”，转让2成虫9 -12μL下降18毫米× 18毫米的盖玻片上的M9或蛋清缓冲区。 </li><li>用针（我们使用27G1 / 2针）或手术刀释放胚胎切割蠕虫开放。试图切断蠕虫的中间。 </li><li>降低到18 × 18毫米，含胚胎盖玻片琼脂糖垫（琼脂面朝下）。修剪琼脂用刀片盖玻片边缘延伸。 </li><li>使用画笔或细木棍，适用于熔化的凡士林盖玻片四边密封盖玻片。 </li></ol>替代琼脂坐骑都挂滴23，如果胚胎受到的压力（即如果蛋壳不形成正确） 。影像也可以在子宫内 ，按照受精或的早期事件，如减数分裂。蠕虫应与左旋咪唑24麻醉。为了增加胚胎数量，从一个特殊的发展阶段几个蠕虫可能被解剖，在同一时间，轻轻地用细尖吸管或睫毛刷在一起定位的胚胎或经口吹打。 4D Nomarski DIC（微分干涉对比）电影<ol start="8"><li>开启奥林巴斯BX61显微镜。由于相机其他设备发出的电磁波非常敏感，它是打开最后，汞灯泡（如果必要的绿色荧光蛋白或荧光）的第一。一旦一切都开始微经理。 DIC或荧光成像选择适当的配置文件。主要的区别是，DIC文件不包括荧光快门的控制。每个文件还可以选择适当的过滤立方体和摄像机的增益设置。 </li><li>胚胎使用10倍的目标显微镜。为了得到成像领域内的多个胚胎，幼胚组寻找附近的蠕虫病毒机构。避免胚胎内最好的图像蠕虫。 </li><li>使用软件切换到一个更高的放大倍率（40倍到100倍）和分辨率（NA为0.9至1.4）的目标成像。 </li><li>调整Nomarski DIC的光学（以下网站包含有用的解释DIC的光学和术语; <a href="http://www.olympusmicro.com/primer/techniques/dic/dichome.html">http://www.olympusmicro.com/primer/techniques/dic/dichome.html</a> http://microscopy.berkeley.edu/Resources/instruction/DIC.html） <ol class="lalpha"><li>在我们的奥林巴斯BX61的重要组成部分是： <ol><li>偏光片 - 滑块下方的冷凝器</li><li>冷凝器Wollaston棱镜 - 炮塔上的冷凝器，略低于阶段。 </li><li>目的Wollaston棱镜 - 滑块下面的过滤器的多维数据集</li><li> 第二偏光器（仪） -过滤器多维数据集在DIC的位置</li></ol></li><li>开关滤波器立方体DIC的位置在光线的路径（软件应该做的启动）分析仪（第二偏光镜） 。 </li><li>确保这两个偏振片是正确对齐（交叉）。当两个沃拉斯顿棱镜光路的视野应该是黑暗的。如果有必要，旋转下面的冷凝器，偏光片，直到这是正确的。 </li><li>插入的目标（上）Wollaston棱镜光路。 </li><li>冷凝器的旋转炮塔，选择Wollaston棱镜您使用的是相匹配的目标。 </li><li>低于这个炮塔调整的滑块，以配合镜头的NA。 </li><li>聚焦最佳克勒照明冷凝器。 </li><li>调整上沃拉斯顿PRISM，以获得最佳的对比度滑块旋转旋钮。 </li><li>软件启动控制的其他设置包括摄像机的增益（设置为0），并切换为8位，16位TIFF文件保存文件。 </li></ol></li><li>调光的水平，以良好的形象。 （通常使用4.7伏）（通过软件控制）。我们宁愿照亮样本，所以，最亮的像素略低于饱和水平。 良好的DIC的光学，结果在一个三维的外观，在胚胎的卵黄颗粒清楚脱颖而出形象。另外这可谓仿佛照亮样品，似乎从某个角度照射，使样品的一面（或样品中的功能）亮起明亮，对面的阴影，并出现较深。 </li><li>选择利息率（ROI）的地区，包括您想要的图像的胚胎，点击设定投资回报率，以减少窗口的按钮。 </li><li>打开的多维采集窗口。选择的Z堆栈（片）和时间点。 </li><li>使用显微镜对焦旋钮找到胚胎的底部（去年一些重点蛋黄颗粒焦平面）。点击选择底部。重复，以确定和设置胚胎顶部。我们通常使用1微米的步长和结束〜20焦平面。我们一般在每个时间点收集多个焦平面，因为胚胎和细胞都比较厚。此外，特别是当细胞开始划分在不同的方向，他们可以取代其他细胞在胚胎周围原焦平面或移动。如果您的显微镜没有对焦马达，您可以手动调整焦距按照您感兴趣的细胞或过程。 </li><li>输入所需的时间点和时间间隔的数目。我们通常使用15秒的时间间隔，并设置了500个时间点的最大。我们开始有更多的时间比我们需要停止收购时所需。如果你是多个焦平面成像，确保有足够的时间获取所有时间点之间的时间间隔焦平面。 </li><li>选择或创建一个位置来保存数据。确保该软件设置为只显示最后拍摄的影像。给文件命名，单击“获取”。监视器前几个时间点的自动图像采集，以确保其正常运行。 </li><li>单击“停止”当你想停止采集，切换到10倍的目标，并准备下一张幻灯片。 </li><li>当做成像光路中删除DIC的组件。必要时关闭目标的清洁油。打开关闭相机开始的一切。 </li></ol> GFP的电影<ol start="20"><li>启动显微镜，上述发现胚胎。使用配置文件，其中包括荧光快门的软件控制。 </li><li>确保不上DIC棱镜（滑块）在光路。 </li><li>开关滤波器立方体FITC / GFP（启动时自动设置） </li><li>摄像机的增益为255，图像文件更改为16位TIFF（在这个配置文件启动时自动设置）。 </li><li>关闭白光。 </li><li>点击预览图像实时成像。快速地工作，以尽量减少光线照射。另外，使用捕捉图像收集单一的形象。 </li><li>调整汞灯泡的功率/中性密度滤光片，曝光长度，获得了良好的形象。在一般情况下，你应该使用最小的光到达样品，以获取您想要的数据，以减少光损伤的细胞和漂白的荧光信号。 </li><li>设置的时间间隔，时间点的数量和焦平面。我们经常使用5-10秒的时间间隔和1-3焦平面。 </li><li>选择投资回报率和图像采集设置参数如前面描述的步骤13-19。 </li></ol>分析电影<ol start="29"><li>数据将被保存到文件夹与您输入的名称，并包含一系列4D栈每个图像的TIFF文件，文本文件和一个XML文件包含元数据，如暴露水平和时间间隔。 </li><li> ImageJ通过两种方法可以打开TIFF图像。第一（方法一）是最简单的，也将读取每个文件的元数据。第二（方法二）能够使用虚拟内存来打开文件，比分配到ImageJ的内存量，但它不包括元数据。 <ol class="lalpha"><li>利用微管理器插件<ol><li>微观管理是建立在使用ImageJ（将不同，您可能以前安装的任何其他版本，它安装一个新版本的ImageJ），并包括一个插件来打开数据。另外，您可以复制到plugins文件夹的Micro-Manager-1.3/plugins文件夹中的内容为另一个版本ImageJ。 </li><li>转到“插件”菜单，选择微经理，然后开微管理器文件。 </li><li>选择该文件夹包含您要查看的数据，然后单击“确定”。 </li><li>玩通过的Z和T尺寸。 </li></ol></li><li>使用位点plugiN打开BioFormats进口商的电影。 <ol><li>传输XML和TXT文件，包含TIFF文件的文件夹，重命名这两个文件的数据集的名称相匹配。 </li><li>在ImageJ插件下拉菜单，选择位点，然后无论是数据的浏览器或生物格式进口商。 </li><li>查找与您的TIFF栈文件夹。点击第一个TIFF文件，然后打开。 </li><li>与Hyperstack栈。根据数据集组织，类似的名称交换尺寸：选择一组文件，并打开所有系列。在内存管理：选择：使用虚拟堆栈（特别是对于大型数据集）。点击ok </li><li>该插件将确定数据集的尺寸（＃时间点和焦平面），并显示&lt;&gt;括号之间的范围。单击确定或改变的时间点和/或焦平面打开数量。 </li><li>确认每个系列对应的维度。 </li><li>玩通过的Z和T尺寸。 </li></ol></li></ol></li></ol>代表性的成果<img src="/files/ftp_upload/2625/2625fig1.jpg" alt="图1" /> 从图1。Nomarski DIC的电影剧照说明年初三期间正常的细胞活动线虫胚胎发育：原核迁移（0-5:45），非对称第一师（6:45-14:45）和异步第二师（14:45-27:00） 。数据收集与60X 1.35 NA透镜。 20间隔1微米的焦平面收集每15秒与40微秒曝光。图像旋转，使后的权利和腹侧下降。比例尺代表10微米。 <img src="/files/ftp_upload/2625/2625fig2.jpg" alt="图2" /> 图2说明的GFP标记的染色体乘客复杂亚基（AIR - 2）在染色体上的早期后期（A至C），然后在后期出现的主轴midzone上，直到目前，从中期的动态变化从一个荧光电影剧照胞质完成（C至F）。数据收集与60X 1.35 NA透镜。一个单一的焦平面收集每隔10秒与50微秒曝光。图像旋转，因此，后是正确的。比例尺代表10微米。 电影1 Nomarski电影野生型的胚胎 。 电影相应的图1。后路是右下角和腹侧向左下方。显示一个单一的原始数据集焦平面。图片收集每15秒和回放设置在每秒14帧。 <a href="http://www.jove.com/files/ftp_upload/2625/Movie1.mp4">点击这里观看视频</a> 野生型的胚胎表达GFP的荧光电影电影2：AIR - 2。 对应图2的电影。后路是向右上方。收集作为一个单一的焦平面的电影。图片收集每10秒和回放设置在每秒14帧。 <a href="http://www.jove.com/files/ftp_upload/2625/Movie2.mp4">点击这里观看视频</a>

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M., Campbell, J. M., White, J. G., Spang, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+actin+cytoskeleton+and+homotypic+membrane+fusion+in+ER+dynamics+in+Caenorhabditis+elegans.">Involvement of the actin cytoskeleton and homotypic membrane fusion in ER dynamics in Caenorhabditis elegans.</a> Mol Biol Cell. 16, 2139-2153 (2005).</li><li>Bianchi, L., Driscoll, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+embryonic+C.+elegans+cells+for+electrophysiological+and+pharmacological+analyses.">Culture of embryonic C. elegans cells for electrophysiological and pharmacological analyses.</a> WormBook. , 1-15 (2006).</li></ol>

居住时间推移成像的一个主要考虑的是保持细胞和有机体的完整性和可行性。 DIC的显微镜提供该样本是不能暴露在紫外线灯光照明光源过热的好处。非常适合DIC的显微镜检测细胞迁移和细胞形态的变化，如胞质，并确定一些亚细胞结构，如纺锤体和核膜。记者蛋白的荧光成像补充额外的亚细胞识别并允许特定蛋白质的可视化DIC的显微镜。为了减少光毒性和损害胚胎在荧光成像危害，我们通常会增加数码相机增益，以抵消减少紫外线灯的照明强度和持续时间。淡淡荧光的转基因记者，去卷积算法重新分配焦光或去模糊算法，以减少背景可以提高拍摄的图像质量。虽然有时是必要的，如共聚焦或多光子系统的先进的显微镜，我们发现，许多荧光记者可以使用此啶显微镜设置，产生高质量的图像进行成像。 微观管理（http://www.micro-manager.org），除了是免费的，直接TIFF格式保存的文件，而不是专有的文件格式，许多商业软件包。这大大简化了我们的数据分析，通过消除需要读ImageJ，Photoshop等图像编辑软件中的图像文件的文件转换步骤。

奥林巴斯BX61显微镜滨松ORCA - ER的相机。 10X计划萤石（NA 0.3）和物镜60X PlanAchromat（NA 1.35） 微经理46年3月1日（http://www.micro-manager.org） 图片J 1.43（http://rsbweb.nih.gov/ij） loci_tools.jar（http://www.loci.wisc.edu） 琼脂糖 - 分子生物学/凝胶电泳级费舍尔Superfrost / PLUS显微镜载玻片（目录＃12-550-15） 实验室磁带（费舍尔目录＃15-901系列） 18毫米× 18毫米＃1.5盖玻片 M9（242 mM的KH 2 PO 4，40毫米的Na 2 HPO 4，g氯化钠9毫米，19毫米NH 4 CL硫酸镁4） （http://130.15.90.245/wormlab_recipe_book.htm）。 蛋缓冲区（118毫米氯化钠，氯化钾48毫米，2毫米氯化钙2，2毫米氯化镁2，25毫米的HEPES，pH值7.3）25。 矿脂刷/棒剃刀刀片热区块解剖显微镜蠕虫22 铂丝挑22 27G1 / 2精密滑翔机针（Beckton Dickinson公司）或用小刀片的手术刀

imaging c. elegans embryos using an epifluorescent microscope and open source software

如染色体组装，分离和胞质的细胞过程，本质上是动态的。时间推移活细胞成像，采用荧光标记的记者蛋白质或微分干涉对比（DIC）显微镜，允许这是从固定样本的分析1,2推断，否则这些动态事件的时间进展检查。此外，研究的细胞过程的发展规定，必须进行时间推移，在开发过程中的一个完整的有机体的实验。 Caenorhabiditis线虫胚胎是透明的光，具有快速，不变与发展计划的一个称为细胞谱系3，从而提供了一个理想的实验模型，为研究细胞生物学4,5 和发展6-9 的问题 。C。 线虫是不容修正，以正向遗传学（基于随机突变10,11）和反向遗传学针对特定的基因（基于RNAi介导的干扰和有针对性的诱变 12-15）的基因操纵。此外，转基因动物可以很容易的创建，以表达荧光标记蛋白质或记者 16,17 。这些特点结合起来，使其易于识别的基本细胞和发育过程的遗传途径调节 体内 18-21 。在这个协议中，我们目前 C.实时成像方法线虫胚胎上使用的复合啶显微镜DIC的光学或绿色荧光。我们展示了现成的显微镜，通常用于固定样本成像，还可以缓解时间推移，使用开放源码软件自动成像过程中的应用分析。

Watch this Scientific Journal Video about Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software at JoVE.com

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

“<em&gt; C。线虫</em&gt;胚胎是一个功能强大的系统研究细胞生物学和发展。我们目前的协议为实时成像的<em&gt; C。线虫</em&gt;利用DIC的光学或使用现成的啶显微镜和开放源码软件的荧光的胚胎。

成像 C。线虫胚胎使用啶显微镜和开放源码软件

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

imaging-c-elegans-embryos-using-an-epifluorescent-microscope-open

Research

JoVE Journal

Biology

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

27.7K 次观看.  University of Michigan. “ C。线虫胚胎是一个功能强大的系统研究细胞生物学和发展。我们目前的协议为实时成像的 C。线虫利用DIC的光学或使用现成的啶显微镜和开放源码软件的荧光的胚胎。

视频: Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

科研

生物学

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

编制果蝇实时成像的胚胎使用悬滴协议

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Assembly, Tuning and Use of an Apertureless Near Field Infrared Microscope for Protein Imaging

This paper aims to instruct the reader in the assembly and operation of an infrared near-field microscope for imaging beyond the diffraction limit.  The apertureless near-field microscope is a light scattering-type instrument that provides infrared spectra at circa 20 nm resolution. A complete list of components and a step-by-step protocol for use is provided.  Common errors in assembly and instrument tuning are discussed.  A representative data set that shows the secondary structure of an amyloid fibril is presented.  

The assembly of a nearfield infrared microscope for imaging protein aggregates is described.

大会，调整和使用蛋白成像Apertureless近场红外显微镜

This paper aims to instruct the reader in the assembly and operation of an infrared near-field microscope for imaging beyond the diffraction limit.  The ...

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages as well as by the availability of genetic mutants and transgenic strains expressing a myriad of fusion proteins1-4. In addition, dynamic processes such as cell division can be viewed using fluorescently labeled reporter proteins. The study of mitosis can be facilitated through the use of time-lapse experiments in various systems including intact organisms; thus the early C. elegans embryo is well suited for this study. Presented here is a technique by which in vivo imaging of sub-cellular structures in response to anoxic (99.999% N2; &lt;2 ppm O2) stress is possible using a simple gas flow through setup on a high-powered microscope. A microincubation chamber is used in conjunction with nitrogen gas flow through and a spinning disc confocal microscope to create a controlled environment in which animals can be imaged in vivo. Using GFP-tagged gamma tubulin and histone, the dynamics and arrest of cell division can be monitored before, during and after exposure to an oxygen-deprived environment. The results of this technique are high resolution, detailed videos and images of cellular structures within blastomeres of embryos exposed to oxygen deprivation.

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Described here is an in vivo technique to image sub-cellular structures in animals exposed to anoxia using a gas flow through microincubation chamber in conjunction with a spinning disc confocal microscope. This method is straightforward and flexible enough to suit a variety of experimental parameters and model systems.

使用时间的推移显微镜可视化缺氧诱导假死<em&gt; C.线虫</em&gt;胚胎

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

线虫趋含量的

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

抗体在染色<em&gt; C。线虫</em&gt;使用“冻结开裂”

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

测量细菌的影响<em&gt; C。线虫</em&gt;行为使用一个鸡蛋保留含量

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

对缺血/再灌注在缺氧，饥饿型号<em&gt; C。线虫</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces photo-bleaching and phototoxic effects compared to confocal microscopy, while providing 3D images with subcellular resolution. Here we present the setup of a light sheet based microscope, which is composed of an upright microscope and a small set of opto-mechanical elements for the generation of the light sheet. The protocol describes how to build, align the microscope and characterize the light sheet. In addition, it details how to implement the method for in toto imaging of C. elegans embryos using a simple observation chamber. The method allows the capture of 3D two-colors time-lapse movies over few hours of development. This should ease the tracking of cell shape, cell divisions and tagged proteins over long periods of time.

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

This protocol describes the setup of a light sheet microscope and its implementation for in vivo imaging of C. elegans embryos.

设置简单的轻表为显微镜<em&gt;在托托</em的&gt;成像<em&gt; C。线虫</em&gt;发展

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces ...

Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators.
Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software1&#160;to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.

Presented is a flexible informatics workflow enabling multiplexed image-based analysis of fluorescently labeled cells. The workflow quantifies nuclear and cytoplasmic markers and computes marker translocation between these compartments. Procedures are provided for perturbation of cells using siRNA and reliable methodology for marker detection by indirect immunofluorescence in 96-well formats.

工作流的高含量，荧光标记的万能工具显微镜数据，支持开源软件的单个细胞定量

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly ...