Sequential immunofluorescence staining and image analysis for detection of large numbers of antigens in individual cell nuclei
Sequential immunofluorescence staining and image analysis for detection of large numbers of antigens in individual cell nuclei
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DOI：
10.1002/cyto.10026
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发表时间：
2002-01-01
期刊：
影响因子：
--
通讯作者：
Zetterberg, A
中科院分区：
文献类型：
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作者：
Wählby, C;Erlandsson, F;Zetterberg, A
背景资料：通过免疫组化染色显示一种以上的抗原对于探索功能意义的空间和时间关系通常是期望的，甚至是必要的。以前提出的染色协议已被限制到可视化的三个或四个antigens.Methods：免疫荧光染色进行福尔马林固定的组织切片和细胞培养。使用数字成像荧光显微镜记录染色材料的图像。随后使用变性和洗脱技术的组合去除一抗和二抗以及荧光团。去除荧光染色后，进行新的免疫荧光染色，使一组新的抗原可视化。该程序重复多达三次。为了对图像数据进行有效和客观的分析，提出了一种结合图像分割、数据提取和细胞分类的图像配准方法。结果：结果表明，在许多情况下，免疫荧光染色可以重复去除，而不会对样品的抗原性产生重大影响。因此，可以使用顺序免疫荧光染色和所述图像分析技术半定量地测量每个细胞中至少六种不同抗原的浓度。通过所提出的方案，可以在单个样品中可视化的抗原的数量显著增加。Cytometry，47：32-41，2002. (C)2001 Wiley-Liss，Inc.
Background: Visualization of more than one antigen by multicolor immunostaining is often desirable or even necessary to explore spatial and temporal relationships of functional significance. Previously presented staining protocols have been limited to the visualization of three or four antigens.Methods: Immunofluorescence staining was performed both on slices of formalin-fixed tissue and on cells in culture. Images of the stained material were recorded using digital imaging fluorescence microscopy. The primary and secondary antibodies, as well as the fluorophores, were thereafter removed using a combination of denaturation and elution techniques. After removal of the fluorescence stain, a new immunofluorescence staining was performed, visualizing a new set of antigens. The procedure was repeated up to three times. A method for image registration combined with segmentation, extraction of data, and cell classification was developed for efficient and objective analysis of the image data.Results: The results show that immunofluorescence stains in many cases can be repeatedly removed without major effects on the antigenicity of the sample.Conclusions: The concentration of at least six different antigens in each cell can thus be measured semiquantitatively using sequential immunofluorescence staining and the described image analysis techniques. The number of antigens that can be visualized in a single sample is considerably increased by the presented protocol. Cytometry,47:32-41, 2002. (C) 2001 Wiley-Liss, Inc.