同种属来源一抗在乳腺癌量子点多标记荧光染色中的应用

Multi-target quantum dots fluorescent staining of breast cancer using primary antibodies from the same species

目的:利用多色量子点(QDs)对细胞角蛋白(CK)、雌激素受体(ER)和人类表皮生长因子受体2(HER-2)进行间接法荧光染色,探讨同种属来源一抗多标记QDs染色的可行性和应用效果。方法:利用EliVision二步法对28例浸润性乳腺癌组织中CK、HER-2和ER进行免疫组化染色,利用QDs-565、QDs-655和QDs-605对28例浸润性乳腺癌组织中CK、HER-2和ER进行间接法单标记荧光染色和三标记荧光染色,使用装备有CRi Nuance多光谱成像系统的Olympus BX51荧光显微镜采集荧光光谱信息,并从采集的荧光图像中选取单个乳腺癌细胞进行亚细胞定位分析。结果:对于单个靶标而言,免疫组化染色和QDs荧光染色都能较清楚地显示靶标的亚细胞定位和表达情况。三标记荧光染色结果显示,绿色QDs-565标记了主要表达于肿瘤细胞质的CK,呈现出癌巢的轮廓,红色QDs-655和品红色QDs-605分别标记了表达于肿瘤细胞膜的HER-2和细胞核的ER,两种同种属来源的一抗HER-2和ER未发生明显的交叉反应。对三种靶标的亚细胞定位分析显示,胞核中ER的品红色荧光信号与胞膜上HER-2的红色荧光信号和胞质中CK的绿色荧光信号在空间上分离清楚,未发生明显光谱重叠现象。结论:基于多色QDs的间接法多标记荧光染色,通过调整抗体浓度、优化孵育时间、彻底清洗等手段,可较好地避免一抗种属来源相同时的交叉反应,与CRi Nuance多光谱成像系统进行结合,有利于对各个靶标分子进行分离和亚细胞定位,实现更准确的光谱定量分析。

Objective: To discuss the feasibility of using primary antibodies from the same species in the multiple immuno-fluorescence staining method with the fluorescence staining of cytokeratin(CK), human epidermal growth factor receptor-2(HER-2), and estrogen receptor(ER)proteins using multicolor quantum dots(QDs). Methods: Immunohistochemistry staining of CK, HER-2, and ER was performed in 28 invasive breast cancer specimens using the EliVision method. Indirect single-target and three-target fluorescence staining of CK, HER-2, and ER in these specimens using QDs-565,QDs-655, and QDs-605 was also performed. The fluorescence spectrum was collected by the Olympus BX51 fluorescence microscope with the CRi Nuance multispectral analysis system, and the subcellular localization analysis was performed in several breast cancer cells selected from the collected images. Results: The fluorescence staining results indicated that CK was mainly expressed in the tumor cytoplasm, marked by green QDs-565, showing the outline of tumor nests. HER-2 was expressed in the cell membrane, marked by red QDs-655. ER was expressed in the cell nucleus, marked by magenta QDs-605. There was no obvious cross-reaction between two primary antibodies from the same species(HER-2 and ER). The subcellular localization analysis indicated that the magenta fluorescence signal of ER in the nucleus was clearly separated from the red fluorescence signal of HER-2 on the cell membrane and the green fluorescence signal of CK in the cytoplasm. Conclusion: Through adjusting the concentration of antibodies, optimizing the incubation time, and thorough cleaning, the cross-staining of primary antibodies from the same species can be avoided in the indirect multi-target QDs fluorescence staining method. Combining with the CRi Nuance multispectral analysis system, more accurate spectrum quantitative analysis can be achieved.