Yu Xiaotong, Song Yan, Chen Li, Xue Lixiang, Yang Jianling, Huang Chen
Center of Basic Medical Research, Peking University Third Hospital, 49 North Garden Road, Haidian District, Beijing, 100191, China.
Department of Ophthalmology, Peking University Third Hospital, Beijing, China.
BMC Res Notes. 2025 May 27;18(1):235. doi: 10.1186/s13104-025-07301-4.
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.
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.
酪胺信号放大（TSA）诱导的乳白质多重免疫组织化学（mIHC）是一种用于原位检测多种靶蛋白的先进方法。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存在一些局限性，特别是在应用于容易分层的组织时。微波处理可能会损害组织完整性，此外，化学试剂的有效性可能对温度、pH值和浓度的变化敏感。本研究旨在优化专门用于乳白质mIHC方案的抗体剥离策略，解决当前方法的局限性，特别是对于容易分层的组织。
对微波炉辅助抗体去除（MO-AR）、基于化学试剂的抗体去除（CR-AR）以及在50°C（HO-AR-50）和98°C（HO-AR-98）下基于杂交炉的剥离进行评估，结果显示MO-AR和HO-AR-98最有效地去除了一抗和二抗。在小鼠肾脏切片的五色mIHC中，这两种方法都产生了强烈的靶特异性信号。然而，在容易分层的脑组织切片中，与MO-AR相比，HO-AR-98能更好地保持组织完整性，并支持有效的多靶点染色。这些发现建立了一种与基于TSA的乳白质mIHC兼容的新型热化学剥离方法，提高了其在研究和临床诊断中的实用性。
