免疫化学染色图像分析法检测细胞核因子κB

Detection of nuclear factor kappa B by immunochemical staining and image analysis

目的探讨一种简便、安全、可定量的细胞核因子κB(NF-κB)活性的检测方法。方法以晚期糖基化终产物修饰的人血清白蛋白(HSA-AGE)刺激人脐静脉内皮细胞(HUVEC),NF-κB/p65免疫化学染色后,分析细胞NF-κB/p65核/浆的阳性染色比,以经典的凝胶迁徙率泳动分析法作对照。结果HSA-AGE可激活HUVECNF-κB,呈时间、剂量依赖关系,两种方法检测的趋势一致。结论免疫化学染色的图像分析法可用于定量检测NF-κB的活性。

Obj ec tive To find a relatively simple and safe method for quantitative assay of nucle ar factor (NF)-kappa B activity. Methods Immunochemical staining of NF-kappa B/ p65 subunit with its antibody was performed in human umbilical vein endothelial cells stimulated with advanced glycation end products-modified human serum albu min (AGE-HSA). The ratio of p65 subunit staining in the nuclei and cytoplasm wa s determined by means of imaging analysis, and the results were compared with t hose of electrophoretic mobility shift assay (EMSA). Results NF-kappa B could be activated by AGE-HSA in a time-and dose-dependent manner, and the results obt ained from immunochemical staining were consistent with those from EMSA. Conclu sion p65 immunochemical staining and subsequent image analysis is feasib le in the quantitative detection of NF-kappa B activation.