2000 Fiscal Year Final Research Report Summary
Development of immunohistochemistry using confocal microscopy and microinjection method
| Project/Area Number |
09557002
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
General anatomy (including Histology/Embryology)
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| Research Institution | Gunma University |
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Principal Investigator |
TAKATA Kuniaki Department of Anatomy and Cell Biology, Gunma University School of Medicine, Professor, 医学部, 教授 (20129290)
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| Co-Investigator(Kenkyū-buntansha) |
SUZUKI Masakazu Institute for Molecular and Cellular Regulation, Department of Cell Biology, Research Associate, 生体調節研究所, 助手 (60280913)
SUZUKI Takeshi Institute for Molecular and Cellular Regulation, Department of Cell Biology, Research Associate, 生体調節研究所, 助手 (00261868)
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| Project Period (FY) |
1997 – 2000
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| Keywords | confocal microscopy / micro-injecion / nuclear counter-staining / immunofluorescence / aquaporin / glucose transporter / salivary glands / plasma membrane |
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| Research Abstract |
In the confocal microscopic examination of biological specimens, localization of nuclei gives important information. We screened fluorescent dyes, originally developed for nucleic acids in gel, for the use in multi-fluorescence microscopy. Among dyes tested SYBR GreenI, SYTOX Green, Pico Green are best suited for green fluorescence, YO-PRO-3 for red fluorescence, and TO-PRO-3 for far red fluorescence. Use of these dyes for nuclear-counter staining in salivary glands proved to give superior confocal images, suitable for the reconstruction of the glands. Localization of aquaporin 5 was clear demonstrated in these complex acinus and duct systems. Nuclear fluorescent counterstaining is also useful in determining the localization of channels and transporters in cultured epithelial cells. For the introduction of exogenous genes into cells, we used microinjection and lipofection methods We transiently expressed GLUT4 glucose transporter in cultured cells, and its immunolocalization and translocation to the plasma membrane was monitored with conventional and confocal fluorescence microscopes. We also used green-fluorescen-protein-tagged GLUT4 and its localization was analyzed. Introduction of exogenous genes, followed by three-dimonsional confocal image analyses proved to be a very convenient and powerful tool in the identification of gene product expressed in the cells.
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