免疫荧光双重标记检测奶牛乳腺整联蛋白α5、β1亚基表达

Detection of integrin subunits α5 and β1 by double immunofluorescence labeling staining of cow mammary gland

屠宰健康荷斯坦奶牛,制备乳腺组织冰冻切片,免疫荧光双重标记检测,比较单克隆和多克隆α5、β1亚基一抗的特异性(FITC标记二抗),用SMMHC多克隆一抗,TRITC标记二抗特异标识肌上皮细胞来辅助定位靶信号,Hoechst33258复染细胞核,在激光共聚焦显微镜下连续断层扫描并拍照。结果表明,借助对肌上皮细胞和细胞核染色定位清晰可见乳腺结构,α5、β1亚基主要分布在导管和腺泡腔上皮细胞膜、外围肌上皮细胞膜和间质中的成纤维细胞膜上,前者在面向腔和靠近基膜的细胞顶侧和基底侧表达均较强,后者仅在基底侧细胞膜上。对于具有极性分布的细胞膜抗原单克隆一抗定位更准确。α5、β1亚基定位在相同区域表明,α5β1异二聚体具有在导管和腺泡基底侧极性分布的表达特点,α5亚基在面向导管和腺泡腔的上皮细胞顶膜上表达,而β1亚基不表达或弱表达,显示该区域存在非α5β1异二聚体形式的α5亚基。

We aimed to investigate expression of integrin subunits α5 and β1 proteins on the cell surface by immunofluorescence analyses in mammary gland frozen tissue section from healthy Holstein cow. The slices were stained with anti-integrin polyclonal antibodies compared with monoclonal antibodies, and then, incubated with IgG labeled by FITC. A sequential double staining of myoepithelial cells incubated with polyclonal antibodies of anti-SMMHC and IgG labeled by TRITC was used to assist positioning. Hoechst 33258 conterstained cell nuclei blue. A continuous layer scanning of the slices and image overlapping were performed under laser confocal microscope. Results showed that blue signal in the nucleus and red signal in myoepithelial cells made green fluorescence distinctly in the membrane of luminal epithelial cells of ducts and alveoli, surrounding myoepithelial cells, and stromal flbroblast. Integrin α5 stained both in apical and basal membrane, whereas integrin β1 only in basal/lateral membrane. Immunofluorescence double labeling with monoclonal antibodies was a favorite for uniform distribution membrane antigen. As integrin α5 and β1 both presented in basal membrane, it implies that similar polarity of α5β1 heterodimer expression. Integrin α5 detected in apical membrane wasn＇t in the form of a heterodimer obviously where β1 integrin could not be detected.