| Budget Amount *help |
¥3,940,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥240,000)
Fiscal Year 2007: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2006: ¥2,900,000 (Direct Cost: ¥2,900,000)
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| Research Abstract |
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.
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