蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎
Summary
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。
Abstract
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。
Introduction
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>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>）。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。
Protocol
Representative Results
Discussion
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?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Embryo collection | |||
| Blunt probe for plug checking | Roboz | RS-9580 | |
| Forceps | Roboz | RS-4978 | |
| Surgery scissors | Roboz | RS-5910 | |
| Glass Pasteur pipettes | Fisher | 13-678-20C | |
| Pre-assembled aspirator tube & mouthpiece | Sigma | A5177 | |
| Longer rubber tubing | Fisher | 14-178-2AA | |
| M2 | Millipore | MR-015-D | |
| FHM | Millipore | MR-024-D | |
| Acid Tyrode's solution | Millipore | MR-004-D | |
| Penicillin/Streptomycin | Gibco | 15140 | |
| Bovine Serum Albumin | Sigma | A9647 | |
| 4-well plates | Nunc/Thermo-Fisher | 12-566-300 | |
| Name | Company | Catalog Number | Comments |
| Immunofluorescence | |||
| 96-well U-bottom plates | Fisher | 14-245-73 | |
| Triton X-100 | Sigma | T8787 | |
| Glycine | Sigma | G7403 | |
| Horse serum | Sigma | H0146 | |
| Primary antibodies | Concentration | ||
| CDX2 | Biogenex | AM392-5M | 1:200 |
| GATA6 | R&D | AF1700 | 1:100 |
| GATA4 | Santa Cruz | sc-9053 | 1:100, 10 min fixation |
| GATA4 | Santa Cruz | sc-1237 | 1:100, overnight fixation |
| SOX17 | R&D | AF1924 | 1:100 |
| Nanog | ReproCELL | RCAB0002P-F | 1:500 |
| OCT4 | Santa Cruz | sc-5279 | 1:100, 10 min to overnight fixation |
| DAB2 | BD | BD-610464 | 1:200 |
| Secondary antibodies | Life Technologies | Various | 1:500 |
| Hoechst 33342 | Life Technologies | H3570 | |
| Name | Company | Catalog Number | Comments |
| Imaging | |||
| 35 mm glass-bottom dishes | MatTek | P35G-1.5-14-C | |
| Name | Company | Catalog Number | Comments |
| Segmentation | |||
| Computer running 64-bit Windows OS | n/a | Verify minimal system requirements at http://katlab-tools.org and in Lou et al., (2014) Stem Cell Reports | |
| MATLAB (software) | Mathworks | n/a | |
| MINS (software) | Free | n/a | http://katlab-tools.org |
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