【摘要】 目的:探讨两种盖玻片制备的细胞爬片对激光共聚焦扫描显微镜(Confocal laserscanning microscopy,CLSM)采集免疫荧光图像效果的影响。方法:以适用于CLSM的专用盖玻片(A片)和组织病理学通用的普通盖玻片(B片)制备肾小管上皮细胞NRK52E的细胞爬片,分别进行胞浆蛋白转录激活因子3(STAT3),胞膜蛋白上皮型钙黏附蛋白(E-cadherin)及细胞骨架蛋白纤维状肌动蛋白(F-actin)的免疫荧光染色,在CLSM下以相同设置采集荧光图像并观察结果的异同。结果:CLSM图像显示,A片中胞浆蛋白STAT3弥散分布于胞浆,胞膜蛋白E-cadherin沿细胞连接处连续线状分布,细胞骨架蛋白F-actin向细胞表面伸出毛刺状突起的丝状伪足,均真实反映了蛋白的全部分布;而B片中胞浆蛋白STAT3弥散分布于胞浆,胞膜蛋白E-cadherin在细胞连接处呈不连续的点、短线状分布,细胞骨架蛋白F-actin未显示向细胞表面伸出的丝状伪足,不能完全真实反映蛋白的全部分布。结论:普通组织病理盖玻片用于CLSM的荧光图像观察,有时会使研究工作者在分析荧光图像时得出错误的结论,因此要选择适合CLSM的免疫荧光专用盖玻片,使实验条件趋于标准化。更多还原

【Abstract】 Objective: To investigate the effects of two different types of coverslips on the immunofluorescence imaging with confocal microscopy.Methods: Both Coverslip A,which was the specified one for confocal microscopy,and Coverslip B,which was the common one used in routine histopathology,were applied for construction of cell-seeded coverslip for NRK52E cells.Protein STAT3,E-cadherin and F-actin,which located in cell cytoplasm,membrane and cytoskeleton,respectively,were detected with immunofluorescence staining.The images were collected by confocal microscopy with the same settings.Results: There was no difference in immunofluorescence pattern of cytoplastic protein STAT3 between images from Coverslip A and Coverslip B.Continuous linear distribution of E-cadherin on cytomembrane was observed in images from Coverslip,while dot-like or discontinuous linear pattern of E-cadherin was observed in images from Coverslip B.Filopodia like protrusions of F-actin on the cell surface were observed in images from Coverslip A,while no filopodia of F-actin was observed in images from Coverslip B.Conclusion: Specialized coverslip for confocal microscopy is necessary in immunofluorescence imaging in preventing misleading results.更多还原