| Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement
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Author:
Date:
2016-08-05
[Abstract] The mitochondrial pathway of apoptosis involves a complex interplay between dozens of proteins and lipids, and is also dependent on the shape and size of mitochondria. The use of cellular models in past studies has not been ideal for investigating how the complex multi-factor interplay regulates the molecular mechanisms of mitochondrial outer membrane permeabilization (MOMP). Isolated systems have proven to be a paradigm to deconstruct MOMP into individual steps and to study the behavior of each subset of MOMP regulators. In particular, isolated mitochondria are key to in vitro studies of the BCL-2 family proteins, a complex family of pro-survival and pro-apoptotic proteins that directly control the mitochondrial pathway of apoptosis (Renault et al., 2013).
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[摘要] 凋亡的线粒体途径涉及数十种蛋白质和脂质之间的复杂相互作用，并且还依赖于线粒体的形状和大小。在过去的研究中使用细胞模型不是理想的调查如何复杂的多因素相互作用调节线粒体外膜透化（MOMP）的分子机制。分离系统已被证明是将MOMP解构成各个步骤并研究每个子集的MOMP调节剂的行为的范例。特别地，分离的线粒体是BCL-2家族蛋白的体外研究的关键，BCL-2家族蛋白是直接控制凋亡的线粒体途径的促存活和促凋亡蛋白的复合家族（Renault > et al 。，2013）。
在这个协议，我们描述三个补充程序用于实时调查使用孤立的线粒体MOMP调节器的影响。第一种方法是"肝线粒体分离"，其中从小鼠中分离肝脏以获得线粒体。 "用JC-1和大小分级分离的线粒体标记"是描述标记，按大小分级并标准化线粒体亚群的方法的第二个方法。最后，"实时MOMP测量"协议允许在孤立的线粒体上实时跟踪MOMP。上述程序用于在体外确定线粒体膜形状在分离的细胞和分离的线粒体水平上的作用（Renault等人，2015）。
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| Morphological Quantification of Nuclei and Mitochondria in Serial Block-face Scanning Electron Microscopy Images
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Author:
Date:
2015-09-20
[Abstract] Serial Block-face Scanning Electron Microscopy (SBF-SEM or 3D-EM) is a powerful tool to study biological structure in ultrastructural level. Quantification of cellular ultrastructure is useful to providing biological information. This technique requires not only high quality of tissue fixation and ideal sample embedding to preserve structures, but also delicate 3D image scanning and post-processing of images. We have adapted previous method to optimize the EM technique to detect and study cellular ultrastructure. Here we present the method to embed samples for 3D-EM technique and to quantify the morphological parameters of nucleus and mitochondria.
Part I. Tissue embedding for 3D-EM images
[摘要] 串行块面扫描电子显微镜（SBF-SEM或3D-EM）是研究超微结构水平生物结构的强大工具。 细胞超微结构的定量对于提供生物信息是有用的。 这种技术不仅需要高质量的组织固定和理想的样品嵌入以保存结构，而且还需要精细的3D图像扫描和图像的后处理。 我们改编以前的方法优化EM技术检测和研究细胞超微结构。 在这里我们提出嵌入样品的3D-EM技术和量化的核和线粒体的形态参数的方法。
第I部分：3D-EM图像的组织嵌入
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