Application of SDS-FRL for Quantitative Localization of Brain Molecules

在后基因组时代，构成生物体的大多数蛋白质都已被鉴定出来，需要回答的问题之一是，每个物种的特定蛋白质种类和数量在特定细胞类型及其亚细胞结构域中表达。这些信息对于理解活细胞中所涉及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，常规免疫电子显微镜（包埋前和包埋后免疫标记）已被广泛使用。虽然这些技术，特别是与免疫金颗粒，提供了精确的细节分子定位在不同的亚细胞结构域，可靠的定量免疫反应性往往受到阻碍的低灵敏度和有限的可及性的抗体埋藏在组织中的靶分子。1995年，Fujimoto开发了一种划时代的质膜分子定位技术，称为SDS消化冷冻断裂复制标记（SDS-FRL），通过该技术，质膜上的分子可以被抗体直接接近，并以高灵敏度和高分辨率的二维方式可视化。在本研究项目中，我对该技术进行了一些改进，以建立适用于脑组织神经科学研究的定量定位方法。通过结合这些努力的成果，我在一些原始论文中建立并介绍了SDS-FRL的现行方案，这些方案可以根据各种目的和其他组织进行进一步修改，以充分发挥这种强大技术的潜力。

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.