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Development of immunohistochemistry using confocal microscopy and microinjection method

使用共聚焦显微镜和显微注射方法开发免疫组织化学

基本信息

批准号：
09557002
负责人：
TAKATA Kuniaki
金额：
$ 7.55万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 2000
项目状态：
已结题

项目摘要

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.

在生物标本的共聚焦显微镜检查中，细胞核的定位提供了重要的信息。我们筛选了荧光染料，最初是为凝胶核酸开发的，用于多荧光显微镜。在测试的染料中，SYBR GreenI, SYTOX Green， Pico Green最适合绿色荧光，YO-PRO-3适用于红色荧光，TO-PRO-3适用于远红色荧光。使用这些染料对唾液腺进行核反染色证明可以获得较好的共聚焦图像，适合于腺体的重建。水通道蛋白5的定位在这些复杂的腺泡和导管系统中得到了明确的证明。核荧光反染色也可用于确定培养上皮细胞中通道和转运体的定位。为了将外源基因导入细胞，我们采用显微注射和脂肪转染的方法，在培养的细胞中瞬时表达GLUT4葡萄糖转运蛋白，并在常规和共聚焦荧光显微镜下监测其免疫定位和向质膜的易位。我们还使用绿色荧光蛋白标记的GLUT4进行定位分析。引入外源基因，然后进行三维共聚焦图像分析是鉴定细胞中表达的基因产物的一种非常方便和强大的工具。