Optimization of a thermochemical antibody stripping method for enhanced tyramide signal amplification-based opal multiplex immunohistochemistry in fragile tissues.

IF 1.6 Q2 MULTIDISCIPLINARY SCIENCES

BMC Research Notes Pub Date : 2025-05-27 DOI:10.1186/s13104-025-07301-4

Xiaotong Yu, Yan Song, Li Chen, Lixiang Xue, Jianling Yang, Chen Huang

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引用次数: 0

Abstract

Objective: Tyramide signal amplification (TSA)-induced Opal multiplex immunohistochemistry (mIHC) represents an advanced methodology for the in-situ detection of multiple target proteins. A critical aspect of this technique is the complete removal of primary and secondary antibodies to prevent signal cross-reaction. However, current antibody stripping methods exhibit several limitations, particularly when applied to tissues prone to delamination. Microwave treatment may compromise tissue integrity, additionally, the effectiveness of chemical reagents can be sensitive to variations in temperature, pH, and concentration. This study aims to optimize antibody stripping strategies specifically for use in Opal mIHC protocols, addressing limitations of current methods which particularly for tissues prone to delamination.

Results: Evaluation of microwave oven-assisted antibody removal (MO-AR), chemical reagent-based antibody removal (CR-AR), and hybridization oven-based stripping at 50 °C (HO-AR-50) and 98 °C (HO-AR-98) revealed that MO-AR and HO-AR-98 most effectively removed primary and secondary antibodies. In five-color mIHC on mouse kidney sections, both methods yielded strong target-specific signals. However, in brain tissue sections prone to delamination, HO-AR-98 better preserved tissue integrity compared to MO-AR and supported effective multi-target staining. These findings establish a novel thermochemical stripping method compatible with TSA-based Opal mIHC, enhancing its utility in research and clinical diagnostics.

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脆弱组织中基于强化酪酰胺信号扩增的蛋白石多重免疫组化热化学抗体剥离方法的优化。

目的：酪酰胺信号扩增（TSA）诱导的蛋白石多重免疫组织化学（mIHC）是一种先进的原位检测多种靶蛋白的方法。该技术的一个关键方面是完全去除一抗和二抗，以防止信号交叉反应。然而，目前的抗体剥离方法表现出一些局限性，特别是当应用于容易分层的组织时。微波治疗可能会损害组织的完整性，另外，化学试剂的有效性对温度、pH值和浓度的变化很敏感。本研究旨在优化用于Opal mIHC协议的抗体剥离策略，解决当前方法的局限性，特别是对于容易分层的组织。结果：在50°C （HO-AR-50）和98°C （HO-AR-98）条件下，对微波辅助抗体去除（MO-AR）、化学试剂抗体去除（CR-AR）和杂交烤箱剥离（HO-AR-98）进行评价，MO-AR和HO-AR-98最有效地去除一抗和二抗。在小鼠肾切片的五色mIHC中，两种方法都产生了很强的靶向特异性信号。然而，在易于分层的脑组织切片中，与MO-AR相比，HO-AR-98能更好地保存组织完整性，并支持有效的多靶点染色。这些发现建立了一种与基于tsa的Opal mIHC兼容的新型热化学剥离方法，增强了其在研究和临床诊断中的应用。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

BMC Research Notes Biochemistry, Genetics and Molecular Biology-Biochemistry, Genetics and Molecular Biology (all)

CiteScore

3.60

自引率

0.00%

发文量

363

审稿时长

15 weeks

期刊介绍： BMC Research Notes publishes scientifically valid research outputs that cannot be considered as full research or methodology articles. We support the research community across all scientific and clinical disciplines by providing an open access forum for sharing data and useful information; this includes, but is not limited to, updates to previous work, additions to established methods, short publications, null results, research proposals and data management plans.