Multiplex Staining by Sequential Immunostaining and Antibody Removal on Routine Tissue Sections
Multiplex Staining by Sequential Immunostaining and Antibody Removal on Routine Tissue Sections
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DOI：
10.1369/0022155417719419
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发表时间：
2017-08-01
影响因子：
3.2
通讯作者：
Cattoretti, Giorgio
中科院分区：
文献类型：
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作者：
Bolognesi, Maddalena Maria;Manzoni, Marco;Cattoretti, Giorgio
在同一细胞或组织切片中进行多重免疫染色原位标记引起了人们极大的兴趣。所提出的方法包括使用标记的一抗、荧光染料的光谱分离、荧光团或色原的漂白、先前抗体层的封闭，所有这些都以各种组合的方式进行。该技术推广的主要障碍是定制抗体和仪器的高成本、低通量以及专业技能或设施的稀缺。我们已经验证了一种基于常见一抗和二抗以及广泛可用的荧光图像扫描仪的方法。在应用另一种染色之前，它需要进行多轮四色间接免疫荧光、图像采集和抗体去除（剥离）。对图像进行数字配准并减去自发荧光。通过二硫键裂解和去污剂或离液盐处理来去除抗体，后者随后进行抗原重折叠。超过 30 种不同的抗体染色剂可应用于来自常规固定和包埋组织的单个切片。这种方法需要对硬件和材料进行适度的投资，并使用免费图像分析软件。常规组织切片上的多重分析是一种高通量工具，可用于原位表征肿瘤细胞、反应性细胞、炎症细胞和正常细胞。 (J Histochem Cytochem 65:431-444, 2017)
Multiplexing, labeling for multiple immunostains in the very same cell or tissue section in situ, has raised considerable interest. The methods proposed include the use of labeled primary antibodies, spectral separation of fluorochromes, bleaching of the fluorophores or chromogens, blocking of previous antibody layers, all in various combinations. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, and scarcity of specialized skills or facilities. We have validated a method based on common primary and secondary antibodies and diffusely available fluorescent image scanners. It entails rounds of four-color indirect immunofluorescence, image acquisition, and removal (stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulfide cleavage and a detergent or by a chaotropic salt treatment, this latter followed by antigen refolding. More than 30 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software. Multiplexing on routine tissue sections is a high throughput tool for in situ characterization of neoplastic, reactive, inflammatory, and normal cells. (J Histochem Cytochem 65:431-444, 2017)