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Aiming at a phylogenetic tree of vertebrate photoreceptor cells on the basis of immunocytochemistry

基于免疫细胞化学的脊椎动物感光细胞系统发育树

基本信息

批准号：
09670001
负责人：
TONOSAKI Akira
金额：
$ 1.98万
依托单位：
Yamagata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

1) Production of monoclonal antibodies against lamprey-retinal homogenate.By this project, 23 new monoclonal antibodies were added to the series of antibodies we precedingly produced. According to their binding sites, the 23 antibodies were classified as follows : (1) Putative photopigment-specific antibodies including long photoreceptor cell (LPC) specific, Fl, Al and short (SPC) -specific, HI ; (2) LPC- and SPC -inner segment-specific, C12, H7, and F91 ; (3) Entire cell-body (LPC and SPC) -specific, G3, 04, and F92 ; (4) Several antibodies which were characterized by their binding to broad areas of glial processes, D2, D5, G3, D1, F11, A3, H6, D12, and A8 ; and (5) Pigment epithelium-specific, A1. In the present project, D2 was selected and investigated particularyin distal. D2 was found to bind with intermediate-sized filaments of Muller cell of the retina, supporting cells of the pineal and parapineal bodies, and ependymal cells bordering the ventricular system. By Western blotting against retinal homogenate, D2 antibody was enhanced by a remarkable band at 76kD.The nature of the protein is recognized by D2 antibody will be subject of the next project.2) Electron microscopy of immunogold binding sites.By freeze replica labeling method, a component of rhodopsin in the form dense IMP gathering more or less with immunogold conjugated H16 antibody. For this method, antibody H16 was most effective, so that it was useful as a standard for the EM immunocytochemistry. For D2, however, was detected on intermediate-sized filaments which were mostly hidden under shadowing deposition.

1)抗七鳃鳗视网膜匀浆单克隆抗体的制备：本项目在已有的单克隆抗体基础上，又增加了23株新的单克隆抗体。根据其结合位点，23种抗体可分为以下几类：（1）推定的感光色素特异性抗体，包括长感光细胞（LPC）特异性抗体，Fl、Al和短感光细胞（SPC）特异性抗体，HI ;（2）LPC-和SPC -内节特异性抗体，C12、H7和F91 ;（3）整个细胞体（LPC和SPC）特异性，G3、04和F92 ;（4）几种抗体，其特征在于它们与神经胶质过程的广泛区域结合，D2、D5、G3、D1、F11、A3、H6、D12和A8 ;和（5）色素上皮特异性，A1。在本项目中，选择D2并特别在远端进行研究。D2与视网膜Muller细胞、松果体和松果体旁支持细胞以及脑室系统边缘的室管膜细胞的中等大小的细丝结合。通过对视网膜匀浆的Western blotting检测，D2抗体在76 kD处有一条明显的增强带，D2抗体识别的蛋白质性质将是下一步研究的课题。2）免疫金结合位点的电镜观察，通过冷冻复型标记法，一种视紫红质成分以密集IMP的形式或多或少地与免疫金结合的H16抗体聚集。对于这种方法，抗体H16是最有效的，因此它可用作EM免疫细胞化学的标准。对于D2，然而，检测到的中等大小的长丝，其中大部分隐藏在阴影下的沉积。