The authors would like to thank Stefano Di Talia, Alberto Puliafito, Venkatraman Seshan and Panagiotis Xenopoulos, for input on data handling, analysis and representation, Berenika Plusa for assistance in the design of the immunofluorescence protocol and antibody testing and members of the Hadjantonakis lab for comments on the manuscript and on the development of this protocol. Work in our lab is supported by the National Institutes of Health R01-HD052115 and R01-DK084391, and by NYSTEM N13G-236.

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The authors declare no conflicts of interest.

本协议描述在植入前阶段的小鼠胚胎进行全挂载免疫荧光定量分析的方法。一个强大的免疫协议22后跟高分辨率，整个安装使用定制的软件4聚焦成像和图像分割。而免疫协议的选择不是关键，我们发现这里呈现22是快速，可靠，并为许多我们已经测试了抗体的提供可靠的信号之一。其它协议可遵循，条件是它们的应用是在整个等效实验一致。虽然许多因素会影响每个单独的实验的实验之间的结果，并直接比较不一定可能的，我们已经发现，在重复追求一致性和执行足够技术重复大大降低可变性。因此，一旦进行了优化，固定和immunolabelin克试剂和方案，以及摄像条件应保持等效实验之间恒定。 1）质量差染色，以暗信号，以及2）不理想的分割，具有很多错误（即，过冲和下分割）:这协议秋季的应用过程中可能出现成两组的主要问题。低质量免疫通常是由于样品的定影不当或不适合于整个安装免疫的抗体。确保PFA是新鲜的（无论是新鲜制备或者从-20ºC解冻RT不超过前一个星期）。由于植入前胚胎的体积小，10分钟内固定PFA应该足够了。然而，我们已经发现某些抗体具有较长的固定时间（见材料），以产生较强的信号。如果先前已经测试了一种抗体，提供了弱信号，尝试不同的固定协议。并非所有的抗体上的强劲执行整个安装IMMunofluorescence和初级抗体的选择是在实验的结果是至关重要的。新抗体应当对胚胎进行测试和最佳浓度根据经验确定。在Materials图表我们提供我们已经测试和经常使用的一些抗体的细节。考虑新的抗体时，请参考发表的文献和制造商的信息。注意，不适合在Western印迹工作的所有抗体整个安装免疫荧光，和必要的免疫荧光的浓度可以幅度更高的高达一个数量级。商业次级抗体通常工作良好开箱，但是，是在利用主 - 辅抗体组合的整个等效实验认为将要被比较一致。 图像分割的质量取决于核染色的质量和在胚胎的ICM的核密度。永远追求光明，尖锐的细胞核，并使用高分辨率物镜（40X或更多）。如果核染色信号为低，则使用新鲜等分试样或一个新批。只要核染色不用于定量，采集参数可以为了记录锐利，明亮的核被调节为每个胚胎。同样地，如果比核染色另一个信道用于分割，该特定信道的信噪比将决定分割的质量。晚期囊胚（E4.0起）在ICM中存在一个非常高的核密度。因此，它是在这些阶段高质量的染色是最关键的。然而，分割的错误会发生经常在这几个阶段。引进核估计直径在敏思管道的图像噪声水平，探索利用"查看"按钮，每一步的结果，直到获得满意的结果调整参数。使用产生最佳分割参数数据处理过程中并手动更正错误。注意，延迟阶段的胚胎（〜E4.5）具有较高的细胞计数和手工数据校正将是费时的。 这个协议的限制由用于图像采集的共焦显微镜系统确定。如所讨论的，使用高放大倍数的物镜，具有工作距离，该距离允许整个胚胎的Z轴成像（ - 直径160微米的着床前的小鼠胚胎可高达150）。同样地，可以检测表位的数量将信道显微镜可以采集一次的次数来确定。使用该协议，除了该DNA三种不同的表位（4个通道中的数量），可以同时在每个样品上标记。确保仪器校准对于每个荧光通道，门控优化和激光输出是一致的。 此处所描述的方法允许的单元位置沿所述分析XYZ轴以及蛋白质表达水平的原位量化为在小鼠胚泡每一个细胞。根据我们的经验，使用其他可用的软件替代方法不能提供高分辨率的水平，在这个协议中采用的管道能（见第4与其他工具的直接比较）。在囊胚的ICM中发现的高核子密度导致其他工具来生成了这么多的错误，需要手动校正的程度，使它们的使用是不切实际的。替代地，手动细胞计数和荧光测量已经先前用于细胞亚群或胚胎10,11,18,19,24的限定区域。然而，所有的荧光通道和单元位置，在所有的XYZ轴，为几十存在于每个胚胎细胞的人工计数和测量会如此费时，这将不允许为哪些我们的协议被设计出来的高通量分析时间。此外，说明书一荧光水平的ssessment容易发生偏差，而管道如这里所描述的允许荧光强度为小区身份的后续判定的客观测量。为了分配小区标识，研究者应当建立规范的准则，所有样品被应用于设置一个特定的实验。考虑到各种因素 - 生物技术 - 可以影响免疫标记的结果，这个标准应该采用适当的控制实验和此前公布的数据尽可能确定。我们设想一个近期，其中的算法可以被设计为自动确定小区标识无论实验。然而，这样的工具将仅来自更广泛的应用和电流的方法，最有可能加上机器学习算法的改进。 最后，鉴于这条管道的简单性和定型吗啉小鼠植入前胚胎的术，该协议是为大量胚胎的分析可轻松扩展，从而使空表型5,6或任何其他实验操作7的高通量分析。最后，虽然当前协议已被设计和植入前小鼠胚胎和ESC集落的分析而优化，我们认为没有理由先验为什么它不能被应用到其他系统具有类似特征-即高核密度。我们鼓励其他国家尝试并适应该协议的其他方案，并为今后的改进提供反馈。

鼠标植入前胚胎是一个范例来研究细胞命运说明书和哺乳动物从头上皮的研究出现和体内的多能性的维持，以及一个模型。哺乳动物发育的胚胎植入前阶段致力于建立三种细胞谱系构成囊胚，即多能性外胚层 ​​- 这引起了大多数体细胞组织和生殖细胞 - 和两个胚谱系，滋养外胚层（TE）和原始内胚层（PRE）（图1A）1,2。这个协议描述的步骤（1）收获并修复植入前阶段的小鼠胚胎，（2）执行免疫荧光标记的感兴趣的蛋白质，（3）进行整个安装使用共聚焦显微镜用z-切片功能和成像（4）执行共聚焦图像和随后的定量图像分析的核分割。这条管道可以牛逼他无偏蛋白水平的测量小区标识的分配来表征原位细胞亚群。这种协议可以在少至3来进行-第4天为一个单一垫料（通常最多10个小鼠胚胎），从胚胎收集到数据分析（图1B）。几个窝的同时分析会增加成像和数据分析时间负担，从而延长了协议的总长度。 植入前阶段的小鼠胚胎是一个听话的实验系统，其中，由于其体积小，定型形态3， 在单细胞分辨率的细胞过程TOTO成像非常适合。开展胚胎的统计相关数字的公正，制度层面的分析，自动的，定量分析的管道是可取的。然而，由于内细胞团的囊胚（ICM）的高核密度（ <st荣&gt;图1A，2D），传统的图像分割平台不能提供足够的精度，以建立一个自动化或半自动化的工作流程。在另一方面，手动分割准确，还不允许细胞和胚胎的大型队列的处理，也不是适合重复性的，公正的决心小区身份的 - 学习发育阶段时，这是特别重要的地方标记模式表达还没有完全解决的（例如，不表现出跨越人口二进制分布）。我们最近开发并验证了小鼠植入前阶段的胚胎，并为实现高精度的小鼠胚胎干细胞（ES细胞）的量身定制的图像分割方法，同时需要很少的用户输入4-8。 这里提出的分析管道围绕基于MATLAB的图像分割工具，模块化交互式核分割（分）4。敏思performs无监督核分割上共焦的Z堆叠的大批量的用户已经建立图片属性的最少数量后，利用图形用户界面（GUI）（表1）4。这条管道已被证明有效的高通量数据对野生型蛋白表达和细胞定位的产生，处理实验和转基因胚胎和胚胎干细胞5-7。在本协议中，我们描述的MIN到植入前阶段的胚胎图像分割中的应用。有关胚胎干细胞的敏思性能的例子请参考4,7。自动核分割步骤显著降低了小区识别处理时的负担，而空间和荧光强度测量允许小区身份的无偏测定和基因表达结构域和细胞位置的三维地图的产生在胚胎（图1C </strong&gt;）。此外，该工作流程的可扩展性使其适用于个别窝的分析，通过实验处理的胚胎，或不同的遗传背景5,6胚胎的大同伙。敏思是免费提供的http://katlab-tools.org（软件需要MATLAB许可证）。 没有开发迄今方法允许对小鼠植入前胚胎蛋白质表达和细胞定位这样深入的数据的生成。所有的尝试到目前为止在量化这些类型的数据已被限制为手动判定和对于不同人群的细胞数的定量在胚胎（或者完全手动，或软件辅助）9-19。 （结合敏思软件）这种方法已经被量身定做和鼠标胚胎植入前胚胎干细胞和测试;不过其上具有高的核密度的其它系统的性能，虽然尚未检验，预计是等价的。

<table><tbody><tr><td>&lt;strong&gt;Embryo collection&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Blunt probe for plug checking</td><td>Roboz</td><td>RS-9580</td><td></td></tr><tr><td>Forceps</td><td>Roboz</td><td>RS-4978</td><td></td></tr><tr><td>Surgery scissors</td><td>Roboz</td><td>RS-5910</td><td></td></tr><tr><td>Glass Pasteur pipettes</td><td>Fisher</td><td>13-678-20C</td><td></td></tr><tr><td>Pre-assembled aspirator tube &amp;amp; mouthpiece</td><td>Sigma</td><td>A5177</td><td></td></tr><tr><td>Longer rubber tubing</td><td>Fisher</td><td>14-178-2AA&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>M2</td><td>Millipore</td><td>MR-015-D</td><td></td></tr><tr><td>FHM</td><td>Millipore</td><td>MR-024-D</td><td></td></tr><tr><td>Acid Tyrode&#039;s solution</td><td>Millipore</td><td>MR-004-D</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140</td><td></td></tr><tr><td>Bovine Serum Albumin&amp;nbsp;</td><td>Sigma</td><td>A9647</td><td></td></tr><tr><td>4-well plates</td><td>Nunc/Thermo-Fisher</td><td>12-566-300&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;Immunofluorescence&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>96-well U-bottom plates</td><td>Fisher</td><td>14-245-73</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>G7403</td><td></td></tr><tr><td>Horse serum</td><td>Sigma</td><td>H0146</td><td></td></tr><tr><td>&lt;strong&gt;Primary antibodies&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Concentration&lt;/strong&gt;</td></tr><tr><td>CDX2</td><td>Biogenex</td><td>AM392-5M</td><td>1:200</td></tr><tr><td>GATA6</td><td>R&amp;amp;D</td><td>AF1700</td><td>1:100</td></tr><tr><td>GATA4</td><td>Santa Cruz</td><td>sc-9053</td><td>1:100, 10 min fixation</td></tr><tr><td>GATA4</td><td>Santa Cruz</td><td>sc-1237</td><td>1:100, O/N fixation</td></tr><tr><td>SOX17</td><td>R&amp;amp;D</td><td>AF1924</td><td>1:100</td></tr><tr><td>Nanog</td><td>ReproCELL</td><td>RCAB0002P-F</td><td>1:500</td></tr><tr><td>OCT4</td><td>Santa Cruz</td><td>sc-5279</td><td>1:100, 10 min to O/N fixation</td></tr><tr><td>DAB2</td><td>BD</td><td>BD-610464</td><td>1:200</td></tr><tr><td>Secondary antibodies</td><td>Life Technologies</td><td>Various</td><td>1:500</td></tr><tr><td>Hoechst 33342</td><td>Life Technologies</td><td>H3570</td><td></td></tr><tr><td>&lt;strong&gt;Imaging&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>35 mm glass-bottom dishes</td><td>MatTek</td><td>P35G-1.5-14-C</td><td></td></tr><tr><td>&lt;strong&gt;Segmentation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Computer running 64-bit Windows OS</td><td></td><td>n/a</td><td>Verify minimal system requirements at http://katlab-tools.org and in Lou et al., (2014) Stem Cell Reports</td></tr><tr><td>MATLAB (software)</td><td>Mathworks</td><td>n/a</td><td></td></tr><tr><td>MINS (software)</td><td>Free</td><td>n/a</td><td>http://katlab-tools.org</td></tr></tbody></table>

quantitative analysis of protein expression to study lineage specification in mouse preimplantation embryos

这个协议提供了一个方法来进行定量，原位单细胞蛋白表达的分析，以研究谱系规范 在小鼠植入前胚胎。描述所需胚胎收集，免疫荧光，在共聚焦显微镜成像，和图像分割和分析的过程。此方法允许在胚胎的所有单元中的多个核标志物的表达和空间（XYZ）的定量的坐标。这需要MINS，专门为着床前胚胎和胚胎干细胞（ESC）的菌落的共焦图像的分析开发出一种图像分割的软件工具的优点。 MINS执行通过X，Y和Z维度的无监督核分割，并以最小的用户输入的所有信道产生在三维空间上单元位置信息，以及核荧光水平。虽然该协议已经为图像的分析而优化植入前阶段的小鼠胚胎的，它可以容易地适应于呈现良好信噪比并且其中高核密度带来了障碍的图像分割（例如，胚胎干细胞中的表达分析的任何其他样品的分析（ESC ）菌落，分化培养中的细胞，其它物种或阶段等 ）的胚胎。

为了便于数据解释和介绍，应注意不要收集和操作过程中损坏的胚胎，使所有的细胞和它们的相对位置可分析图2A - D示出了在具有扩展腔的不同阶段的完整胚泡的例子。如果出现损坏，格外小心，应注意分析和解释结果时。 使用该协议生成的数据的质量和可靠性依赖于固定的质量和用于检测所关注的蛋白质的抗体的信噪比。总是用新鲜固定液和测试新的抗体，以适当的阳性和阴性对照，在开始一组实验之前， 图2A示出良好的抗体的例子为一些核蛋白质。 图2B示出为例为细胞质蛋白（DAB2）良好的抗体即图2C示出一个坏染色，具有低信噪比，其中所述样品固定为仅10分钟的一个例子。在这种情况下，用于GATA4的抗体（Santa Cruz，SC-1237）需要O / N固定提供了有力的信号（见24固定O / N和沾有这种抗体胚胎的情况下）。后处理过程中增加的信号电平揭示了一个非常嘈杂的形象。相比之下用于图2A（SC-9053）抗GATA4仅提供10分钟的固定后高信噪比。在材料部分提供了这些细节和其他强大的抗体。 一个良好的MINS分割的限制因素是:a）用于成像的物镜的倍率，B）的Z分辨率和c）的核染色的质量。 图2D示出了具有OI成像胚胎的例子升浸泡40倍物镜与1.30 NA和0.21毫米WD。中间面板显示其中单个核和它们之间的边界可以被区分的ICM的倍率。如果不使用DNA染色（即，赫斯特，DAPI，TO-PRO3，YO-YO1 等 ）用于定量荧光值，采集参数可以单独调整，以获得最佳的信号。底部图显示如何更先进的胚胎，较高的核密度，因此较高的分割误差（箭头和箭头）的可能性。 图3显示的错误，MINS可以提交，如检测凋亡细胞核作为活细胞的例子（星号在板图3AA，抗体，广告 ），过分割（箭头和图3AC）或欠分割箭头（图3AD箭头 ）。 图3B示出了根据分割事件中的Z-片的序列，在两个细胞已被确定为一个。注意MINS分割如何进行Z轴顾及到段包含所有切片其中给定细胞存在的体积。 最后，数据分析的细节将取决于问题进行研究，因此，对于分析过程准则不能在这里给出。然而，我们已经发现某些初始数据转换是有用的图4B。 - D显示 了图4A中所示的标记NANOG和GATA6在胚胎的ICM的荧光值的曲线图。图4B示出了从MINS获得的原始荧光值图4C示出了一个对数变换，它从小区分隔值之后的同一数据轴（平方根变换也是可能的，并产生了类似的曲线图）。 图4D示出了（的不足和过度分割手动校正后）自动℃后相同的数据orrecting的值在荧光的Z相关联的衰减，如在该协议的步骤4.5.2.2说明。这个Z-相关的荧光衰减的例子在图4E的图像和4F地块中给出。 图4G显示了数据转换的效果4.5.2.2解释。代表任一外胚层（红，NANOG + GATA6-）或前（蓝色，NANOG-，GATA6 +）同一性的颜色已被添加到情节作为NANOG和GATA6值的函数。在这个特殊的例子中，电池，其中日志[GATA6]的比率/日志[NANOG]&gt; 1.25被认为是预，其中日志[GATA6]的比率/日志[NANOG] &lt;1被认为是EPI细胞，并与一中间的细胞率被认为是未提交（在此情况下，无）。所有数据转换和重复已经于Rstudio已经完成，但是，软件的选择不是关键的，并且将取决于最终用户。 <img alt="图1"的src ="/文件/ ftp_upload / 53654 / 53654fig1.jpg"/&gt; 图1.胚胎位置，时间轴和分析管道。（A）一图（二）小鼠子宫角和早期胚胎发育的阶段，他们应该找到喇叭的区域对齐。 （B）的每个步骤的定时的试验的时间表。的协议和暂停点（蓝色）的步骤表示。 （C）图像采集和使用敏思分析管道的总结。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53654/53654fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53654/53654fig2.jpg" /> 图2.免疫荧光和核密度 （A） 的例子。与核TR测试抗体的免疫良好效果的例子anscription因素。用AlexaFluor 488驴抗小鼠次级抗体，抗GATA6与AlexaFluor 568驴抗山羊检测抗CDX2，抗NANOG与AlexaFluor 647驴抗兔，抗GATA4（圣克鲁斯，SC-9053 ）与AlexaFluor 488驴抗兔和抗Oct4与AlexaFluor 647驴抗小鼠。所有初级抗体都列在材料表 。胞质GATA4的核染色是非特异性的，并且也出现在GATA4空胚胎（未示出）。为胞浆蛋白，DAB2良好的免疫荧光（B）为例。它已被用AlexaFluor 488驴抗小鼠检测。坏免疫的（C）的实施例，具有低信噪比的核因子。 GATA4已检测与山羊提出了一个抗GATA4（圣克鲁斯，SC-1237;而SC-9053（兔抗GATA4）在用（A））和AlexaFluor 568驴抗山羊。 （ <stroNG&gt; D）呈现完好的胚胎具有良好的核染色的例子是如何与胎龄和核密度的增加如何增加误差敏思分割（箭头- oversegmentation -和箭- undersegmentation）。规模= 20微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53654/53654fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53654/53654fig3.jpg" /> 图3.敏思错误的示例。（一）个人Z-部分从一个胚胎顺序显示敏思分割错误的例子。它们仍然确定为核细胞凋亡被标记在图a，b和d以星号（*）。在图b，编号＃39（箭头），尽管很小，并确定相应的核，因此可以向左校正。在C组，ID＃66（箭）跨越两个不同氮uclei，这些反过来又被单独列出（箭头，编号＃65＃54和＃53）。在面板D，两个小区（编号＃63）已经被确定为一个单独的之一（见箭头标记薄边界），因此此记录应被复制用于细胞计数的目的。 （B）的MINS检测沿着Z轴的细胞。图像显示已错误地评价为一个单一（＃123）的两个单元中的Z-片的序列。注意蓝色区域划定核是如何沿着Z.连续<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53654/53654fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53654/53654fig4.jpg" /> 图4.分割和数据转换的例子。（A）染色NANOG和胚泡的ICM细胞GATA6和相同的核分割的快照图像利用胚胎在核染色通道敏思（赫斯特33342）。在由MINS测定的ICM细胞（BD）NANOG和GATA6荧光值描绘为原始数据（B）中，在执行对数转换（C）的后沿Z轴的荧光衰变校正值和自动分配根据校正小区身份后值（D）。 （E - G）沿Z轴的荧光衰减数据修正的例子。 （E）的标记用Hoechst和抗CDX2 + AlexaFluor 488（AF488）胚泡，示出了沿Z轴荧光减少图像（F）沿赫斯特和AF488 Z轴为胚胎包括在（E）中所示的一个的池荧光值的散点图。灰线表示各组的值的回归曲线。 （G）相同的数据，在F中补偿荧光衰减为每个单元后，使用以下因素:Z坐标*线性模型的斜率（见步骤4.5.2.2在协议）。 图 E -克的最初由节点中的作者发表（http://thenode.biologists.com/99problems/discussion/）每个点代表一个细胞。规模=20μm的。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53654/53654fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 用户输入 </td></tr><tr><td>亮度阈值*（方便在昏暗的图像核探测） </td></tr><tr><td>相对X / YZ分辨率</td></tr><tr><td>渠道细分</td></tr><tr><td>核径</td></tr><tr><td>图像噪声水平</td></tr><tr><td> 敏思输出 </td></tr><tr><td>细分Z-堆栈nuclEI标识</td></tr><tr><td>核量</td></tr><tr><td> XYZ坐标每个细胞核</td></tr><tr><td>平均和荧光值的总和，每个荧光通道和细胞核</td></tr><tr><td> 优点 </td></tr><tr><td>整个堆核精确分割（&gt; 85％） - 认识到相邻的Z-部分细胞核的所有出现</td></tr><tr><td>单细胞分辨率</td></tr><tr><td>胚胎批次的无监督分割</td></tr><tr><td>分割管道可以被保存并重新使用或调整为每个特定胚胎</td></tr></tbody></table> 表1.敏思特点 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td rowspan="3">加载图像</td><td>在提示符下，引入的Z轴相​​对分辨率图像。它可以从使用​​默认阅读器或应用以下公式文件元数据来获得:X（或Y）分辨率/ Z轴分辨率（都在微米）。 </td></tr><tr><td>输入的序列号（1 - 5）的信道进行分割。使用用于分割该DNA染色通道将检测所有单元的图像中的，但是，其他信道可以单独地也分段。 </td></tr><tr><td>输入框在（默认1）开始分割。仅适用于具有多个时间框架的文件。 </td></tr><tr><td>提高图像（可选） </td><td>图像的白色和黑色点可以调整，以方便检测。通常降低了白点和再扩展到0 - 255（8位图像）或0 - 4096（12位图像）将会使图像更亮，并促进暗淡核检测。 </td></tr><tr><td rowspan="2">检测核</td><td>选择所估计的核直径（在囊胚通常在30和40像素）。 </td></tr><tr><td>对于噪声图像的噪声水平可以被调整。否则，默认值将被应用。 </td></tr><tr><td>段核</td><td>使用默认参数。 </td></tr><tr><td>分类核</td><td> MINS可以在基于位置植入前胚胎区分内细胞团（ICM）细胞滋养外胚层（TE）。这也可以手动完成，或基于荧光的水平，如果使用的TE标志。 </td></tr><tr><td rowspan="5">导出结果</td><td>选择要产生的文件: </td></tr><tr><td> - 细分覆盖在使用的信道（.TIFF序列文件） </td></tr><tr><td> - 分割只（.TIFF序列文件） </td></tr><tr><td> - 电子表格的ID对检测到的所有细胞中，XYZ坐标，并为每个单元（.csv文件）所有通道荧光水平（平均总和）。 </td></tr><tr><td>所有文件默认情况下出口。文件保存在SAME目录为图片所在。 </td></tr></tbody></table> 表2.敏思分割管道

Watch this Scientific Journal Video about Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos at JoVE.com

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

该协议提出对蛋白质表达的原位分析进行定量，单细胞研究在小鼠植入前胚胎沿袭规范的方法。必要的囊胚的汇编程序，整个安装蛋白的免疫检测，在一个共焦显微镜，和核分割和图像分析的样品的成像进行描述。

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

quantitative-analysis-protein-expression-to-study-lineage

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

10.8K 次观看.  Sloan Kettering Institute. 该协议提出对蛋白质表达的原位分析进行定量，单细胞研究在小鼠植入前胚胎沿袭规范的方法。必要的囊胚的汇编程序，整个安装蛋白的免疫检测，在一个共焦显微镜，和核分割和图像分析的样品的成像进行描述。 

视频: Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

损失 - 和增益的功能角度进行调查研究早期细胞命运决定在植入前小鼠胚胎

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

科研

发育生物学

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

转录组分析]<em&gt;活体</em&gt;生产牛胚胎植入前胚胎采用双色微阵列平台

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

从详细的空间表达图谱的动态表达数据的时间排序

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

利用标准化定量西方印迹技术, 对跨组织和整个开发过程中的蛋白质水平进行可靠比较

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

生物化学

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

半高通量成像方法和数据可视化工具包，用于分析C. elegans胚胎发育

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

小鼠胚胎细胞的多路复用单细胞mRNA测序分析

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

为早期小鼠胚胎测序准备小型RNA库

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

发育（青蛙）胚胎中的细胞谱系引导质谱蛋白质组学

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design ...

化学

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

生物学

用于单细胞测序的原肠胚胚分离和基因分型

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.

作者聚焦：以单细胞分辨率分析谱系特异性突变胚胎的管道

This paper establishes a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

突变全小鼠胚胎的单细胞 RNA 测序：从上胚层到原肠胚形成结束

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states ...