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目的 探讨NMDAr(N- 甲基- D- 天冬氨酸受体)在神经细胞膜表面定位,对比不同成像方法的特点,建立纳米尺度神经细胞膜表面蛋白单分子三维标记的免疫细胞化学新方法。方法 应用原子力显微镜,对分布在云母表面的抗NMDAR1亚单位IgG 葡萄球菌蛋白A 胶体金颗粒免疫标记复合物分子进行扫描,明确其特定的三维结构作为对照标准,然后扫描结合了免疫标记复合物分子的神经细胞膜表面,对表面形貌作出对比后确定目的抗原的定位和复合物三维结构。并与光镜、激光共聚焦、扫描电镜等方法进行对比。结果 在空白云母表面,免疫复合物分子在原子力显微镜下呈现出均匀分散粒径为49nm的特征性球形结构。在神经元表面结合免疫标记物后可以发现有大量的粒径为53nm的球形或双球形(短棒状)结构,颗粒均匀,截面为双峰或单峰。光镜下染色成片状结果,在共聚焦显微镜下荧光呈颗粒点状分布,扫描电镜结果为单个或结合颗粒,缺乏三维成像能力。结论 原子力显微镜下免疫胶体金标记技术可以在纳米尺度对目的膜受体蛋白进行定位和表面三维结构测定。NM- DA受体蛋白可以结合一个或两个胶体金复合物,与传统成像手段相比,原子力显微镜下胶体金标记方法可以对单个膜NMDA受体蛋白抗原进行定位、定性、定量标记测定。
Objective To investigate the localization of NMDAr (N-methyl-D-aspartate receptor) on the surface of nerve cell membrane and to compare the characteristics of different imaging methods to establish a new immunocytochemistry method of single-molecule three-dimensional labeling of membrane proteins on the surface of nanoscale cells. Methods Atomic force microscopy was used to scan the anti-NMDAR1 IgG staphylococcal protein A colloidal gold particle immuno-labeled complex molecules distributed on the surface of mica. The specific three-dimensional structure was defined as a control standard, and then the immunolabeled complex molecule Of the nerve cell membrane surface, the morphology of the surface contrast to determine the purpose of the antigen localization and complex three-dimensional structure. And compared with light microscope, laser confocal, scanning electron microscopy and other methods. Results On the mica surface, the immunocomplex molecules showed a characteristic spherical structure with an average particle size of 49 nm under an atomic force microscope. A large number of spherical or double spherical (rod-shaped) structures with a particle size of 53 nm were found after binding of the immunological markers to the surface of the neurons. The particles were uniform and the cross section was bimodal or singlet. Under the light microscope, the results of the staining were flaky. The fluorescent spots were distributed under the confocal microscope. The results of scanning electron microscopy were single or combined particles, which lacked the ability of three-dimensional imaging. Conclusion Immunogold labeling method can detect the three-dimensional structure of the target membrane protein at the nanoscale. NM-DA receptor protein can bind one or two colloidal gold complexes. Compared with traditional imaging methods, colloidal gold labeling method by atomic force microscopy can locate, qualitatively and quantitatively detect single membrane NMDA receptor protein antigens.