Application of SDS-FRL for Quantitative Localization of Brain Molecules

在后基因组时代，当大多数构成生物体的蛋白质已经被确定时，需要回答的一个问题是，在特定的细胞类型及其亚细胞结构域中表达的特定蛋白质种类和每种蛋白质的数量。这些信息对于理解活细胞的机制是必不可少的。对于需要纳米级分辨率的亚细胞定位，传统的免疫电子显微镜（包埋前和包埋后免疫标记）已被广泛使用。尽管这些技术，特别是免疫金颗粒，已经提供了不同亚细胞区域分子定位的精确细节，但免疫反应性的可靠定量常常受到低灵敏度和抗体对组织中隐藏的靶分子的有限可及性的阻碍。1995年，Fujimoto开发了一种划时代的质膜分子定位技术，称为sds -酶切冷冻断裂复制标记（SDS-FRL），通过这种技术，抗体可以直接接近质膜上的分子，并以高灵敏度和高分辨率的二维方式可视化。在这个研究项目中，我对这一技术进行了一些修改，以建立对神经科学研究有用的脑组织定量定位方法。结合这些努力的成果，我在一些原始论文中建立并介绍了目前的SDS-FRL方案，可以针对各种目的和其他组织进一步修改，以充分发挥这一强大技术的潜力。

One of the questions to be answered in the post-genomic era, at a time when most proteins constituting living organisms have been identified, is what particular protein species and amount of each species is expressed in a particular cell type and in their subcellular domains. Such information is indispensable for understanding mechanisms involved in living cells. For subcellular localization that requires nano-scale resolution, conventional immunoelectron microscopy (pre-and post-embedding immunolabeling) has been widely used. Although these techniques, especially with immunogold particles, have provided precise details of molecular localization in different subcellular domains, reliable quantification of immunoreactivity has often been hampered by low sensitivity and limited accessibility of antibodies to target molecules buried in tissues. In 1995, Fujimoto developed an epoch-making technique for localization of plasma membrane molecules, termed SDS-digested freeze-fracture replica labeling (SDS-FRL), by which molecules on the plasma membrane can be directly approached by antibodies and visualized in a two-dimensional manner with high sensitivity and high resolution. In this research project, I have applied some modifications onto this technique in order to establish quantitative localization method useful for neuroscience research using brain tissue. By combining outcomes from these efforts, I established and introduced current protocol of SDS-FRL in some original papers, which could be further modified for various purposes and other tissues to bring out the full potential of this powerful technique.