Washington University in St. Louis.
Washington University in St. Louis; University of California San Francisco.
J Vis Exp. 2023 Feb 24(192). doi: 10.3791/64881.
Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate identification and localization of proteins, glycans, and small molecules. While this technique is well-established in two-dimensional (2D) cell culture, less is known about its use in three-dimensional (3D) cell models. Ovarian cancer organoids are 3D tumor models that recapitulate tumor cell clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Thus, they are superior to cell lines for the evaluation of drug sensitivity and functional biomarkers. Therefore, the ability to utilize immunofluorescence on primary ovarian cancer organoids is extremely beneficial in understanding the biology of this cancer. The current study describes the technique of immunofluorescence to detect DNA damage repair proteins in high-grade serous patient-derived ovarian cancer organoids (PDOs). After exposing the PDOs to ionizing radiation, immunofluorescence is performed on intact organoids to evaluate nuclear proteins as foci. Images are collected using z-stack imaging on confocal microscopy and analyzed using automated foci counting software. The described methods allow for the analysis of temporal and special recruitment of DNA damage repair proteins and colocalization of these proteins with cell-cycle markers.
免疫荧光是一种广泛应用于高灵敏度和特异性检测靶抗原的技术，能够准确识别和定位蛋白质、糖和小分子。虽然这项技术在二维（2D）细胞培养中已经得到很好的建立，但在三维（3D）细胞模型中的应用却知之甚少。卵巢癌类器官是 3D 肿瘤模型，能够重现肿瘤细胞克隆异质性、肿瘤微环境以及细胞-细胞和细胞-基质相互作用。因此，它们比细胞系更适合评估药物敏感性和功能生物标志物。因此，能够在原发性卵巢癌类器官上使用免疫荧光技术对于了解这种癌症的生物学特性非常有益。本研究描述了在高级别浆液性患者来源的卵巢癌类器官（PDOs）中检测 DNA 损伤修复蛋白的免疫荧光技术。在将 PDO 暴露于电离辐射后，对完整的类器官进行免疫荧光染色，以评估核蛋白作为焦点。使用共聚焦显微镜的 z 堆叠成像收集图像，并使用自动焦点计数软件进行分析。所描述的方法允许分析 DNA 损伤修复蛋白的时间和特殊募集以及这些蛋白与细胞周期标志物的共定位。
