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

1)七鳃鳗-视网膜匀浆单克隆抗体的制备。通过这个项目，我们在之前生产的抗体系列中增加了23个新的单克隆抗体。根据结合位点的不同，23种抗体可分为以下几种：①推测的光色素特异性抗体包括长光受体细胞（LPC）特异性抗体Fl、Al和短光受体细胞（SPC）特异性抗体HI；(2) LPC-和SPC -内段特异性，C12， H7和F91；(3)全细胞体（LPC和SPC）特异性，G3, 04和F92；(4)与D2、D5、G3、D1、F11、A3、H6、D12和A8等神经胶质突起广泛结合的几种抗体；色素上皮特异性，A1。在本项目中，D2被选择和研究，特别是在远端。发现D2与视网膜Muller细胞的中等大小纤维、松果体和顶旁体的支持细胞以及与心室系统接壤的室管膜细胞结合。对视网膜匀浆进行Western blotting， D2抗体在76kD处明显增强。该蛋白的性质是否被D2抗体识别将是下一个项目的课题。2)免疫金结合位点的电镜观察。通过冷冻复制标记法，视紫红质成分以密集的IMP形式或多或少地聚集在免疫金偶联的H16抗体上。在此方法中，抗体H16是最有效的，因此它可以作为EM免疫细胞化学的标准。而D2主要在中等大小的细丝上检测到，这些细丝大多隐藏在阴影沉积下。

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.