A Modified Bleaching Method for Multiplex Immunofluorescence Staining of FFPE Tissue Sections.

IF 1.3 4区 医学 Q3 ANATOMY & MORPHOLOGY Applied Immunohistochemistry & Molecular Morphology Pub Date : 2024-11-01 Epub Date: 2024-10-07 DOI:10.1097/PAI.0000000000001228
Dan Wang, Alison Cheung, Gordon E Mawdsley, Kela Liu, Yulia Yerofeyeva, Kelsie L Thu, Ju-Yoon Yoon, Martin J Yaffe
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Abstract

Multiplex immunofluorescence (mIF) staining plays an important role in profiling biomarkers and allows investigation of co-relationships between multiple biomarkers in the same tissue section. The Cell DIVE mIF platform (Leica Microsystems) employs an alkaline solution of hydrogen peroxide as a fluorophore inactivation reagent in the sequential staining, imaging, and bleaching protocol for use on FFPE sections. Suboptimal bleaching efficiency, degradation of tissue structure, and loss of antigen immunogenicity occasionally are encountered with the standard bleaching process. To overcome these impediments, we adopted a modified photochemical bleaching method, which utilizes an intense LED light exposure concurrent with the application of hydrogen peroxide. Repeated stain/bleach rounds with different antibodies were performed on breast tissue and other tissue sections. Residual signal after conventional bleaching and the modified technique were compared and tissue integrity and antigen immunogenicity were assessed. The modified technique effectively eliminates fluorescence signal from previous staining rounds and produces consistent results for multiple rounds of staining and imaging. With the modified method, photochemical treatments did not destroy tissue sub-cellular contents, and the tissue antigenicity was well preserved during the entire mIF process. Overall processing time was reduced from 36 to 30 hours in an mIF procedure with 8 rounds. With the conventional method, tissue quality was highly degraded after 8 rounds. The new technique allows reduced turn-around time, provides reliable fluorophore removal in mIF with excellent maintenance of tissue integrity, facilitating studies of the co-localization of multiple biomarkers in tissues of interest.

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用于 FFPE 组织切片多重免疫荧光染色的改良漂白法
多重免疫荧光(mIF)染色在分析生物标记物方面发挥着重要作用,可以研究同一组织切片中多种生物标记物之间的相互关系。Cell DIVE mIF 平台(徕卡显微系统公司)采用碱性过氧化氢溶液作为荧光团灭活试剂,依次进行染色、成像和漂白,适用于 FFPE 切片。标准漂白工艺偶尔会出现漂白效率不理想、组织结构退化和抗原免疫原性丧失等问题。为了克服这些障碍,我们采用了一种改良的光化学漂白方法,即在使用过氧化氢的同时使用强 LED 光照射。在乳腺组织和其他组织切片上使用不同的抗体进行重复染色/漂白。比较了传统漂白和改良技术后的残留信号,并评估了组织完整性和抗原免疫原性。改良技术能有效消除前几轮染色产生的荧光信号,并在多轮染色和成像中产生一致的结果。采用改进的方法,光化学处理不会破坏组织亚细胞内容物,组织抗原性在整个 mIF 过程中得到了很好的保存。在 8 轮 mIF 过程中,整体处理时间从 36 小时缩短到 30 小时。而采用传统方法,8 轮处理后组织质量会严重下降。新技术缩短了周转时间,在 mIF 过程中可靠地去除荧光团,并很好地保持了组织的完整性,有助于研究多种生物标记物在相关组织中的共定位。
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来源期刊
Applied Immunohistochemistry & Molecular Morphology
Applied Immunohistochemistry & Molecular Morphology ANATOMY & MORPHOLOGY-MEDICAL LABORATORY TECHNOLOGY
CiteScore
3.20
自引率
0.00%
发文量
153
期刊介绍: ​Applied Immunohistochemistry & Molecular Morphology covers newly developed identification and detection technologies, and their applications in research and diagnosis for the applied immunohistochemist & molecular Morphologist. Official Journal of the International Society for Immunohistochemisty and Molecular Morphology​.
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