不同固定剂在激光共聚焦荧光技术中的效果评价

Evaluation of Different Fixatives in Confocal Immunofluorescence

【目的】探讨3种不同的固定剂在激光共聚焦免疫荧光技术中对信号表达的影响。【方法】对培养的NRK-52E细胞分成胞浆,胞膜及胞核抗原3组,每组用4g/LPFA,甲醇及丙酮分别固定后,完成免疫荧光实验;原代滑膜成纤维样细胞分两组用4g/LPFA固定后分别用TritonX-100和预冷的甲醇进行打孔,完成免疫荧光实验之后,使用Confocal显微镜在条件同等的情况下观察并拍摄荧光图像,以证实不同的固定剂对信号表达的影响。【结果】胞浆蛋白α-SMA用丙酮固定时信号清晰锐利,将Actin蛋白的丝状结构表达得非常充分。胞膜蛋白ZO-1用甲醇固定处理的信号最清晰。胞核蛋白磷酸化的Smad3用4g/LPFA固定时p-Smad3信号与细胞核具有很好的共定位关系。原代滑膜成纤维样细胞用预冷甲醇打孔处理的细胞,其磷酸化的骨架相关蛋白(P-ERM)蛋白信号明显优于用TritonX-100打孔处理的细胞。【结论】针对不同抗原结构特点选用固定剂;有机溶剂固定剂用于细胞支架成分的固定,交联剂固定剂用于膜相关成分的固定,同时使用交联剂固定剂及有机溶剂打孔可较好的保存抗原而获得高质量的荧光图片。

【Objective】 To evaluate the immunostaining effect of three different fixatives by confocal microscopy. 【Methods】 NRK-52E cells were cultured and fixed by 4 g / L PFA, methanol, and acetone, respectively, followed by three different antibodies to determine the representative antigens in membranes, cytoplasm, and nucleus under the confocal microscopy. Primary synovial fiber-like cells were cultured and fixed by 4 g / L PFA, permeabilizing by TritonX-100 and cooled methanol respectively. Immunostaining results of detecting the P-ERM protein were performed by confocal microscopy under the same observing condition. 【Results】 The signals of a cytoplasm skeleton protein and α-smooth muscle actin （α-SMA） showed sharp and clear, when the NRK cells were treated with acetone. A membranes protein, ZO-1 showed best results when the cells were treated with methanol. Compared to other fixatives, the cells fixed with 4 g / L PFA showed the best co-localization results of p-Smad3 protein with nucleus signals. The signals of P-ERM on primary synovial fiber-like cells indicated that it was significantly better when the cell permeabilizing by cooled methanol, comparing with permeabilization by TritonX-100. 【Conclusion】 Organic fixative were applied for fixing the cytoskeleton components and cross-linking fixative were suit for fixing the membrane components. Fixing by crosslinking agents followed by permeabilization of organic solvents can keep the antigens well and therefore could be used to take high quality pictures.