Optimization of a thermochemical antibody stripping method for enhanced tyramide signal amplification-based opal multiplex immunohistochemistry in fragile tissues
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 preve...
Saved in:
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2025-05-01
|
| Series: | BMC Research Notes |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13104-025-07301-4 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849704377190711296 |
|---|---|
| author | Xiaotong Yu Yan Song Li Chen Lixiang Xue Jianling Yang Chen Huang |
| author_facet | Xiaotong Yu Yan Song Li Chen Lixiang Xue Jianling Yang Chen Huang |
| author_sort | Xiaotong Yu |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-e8753aa26d1c4f5baf3f47daf75f142f |
| institution | DOAJ |
| issn | 1756-0500 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Research Notes |
| spelling | doaj-art-e8753aa26d1c4f5baf3f47daf75f142f2025-08-20T03:16:46ZengBMCBMC Research Notes1756-05002025-05-0118111310.1186/s13104-025-07301-4Optimization of a thermochemical antibody stripping method for enhanced tyramide signal amplification-based opal multiplex immunohistochemistry in fragile tissuesXiaotong Yu0Yan Song1Li Chen2Lixiang Xue3Jianling Yang4Chen Huang5Center of Basic Medical Research, Peking University Third HospitalCenter of Basic Medical Research, Peking University Third HospitalCenter of Basic Medical Research, Peking University Third HospitalCenter of Basic Medical Research, Peking University Third HospitalCenter of Basic Medical Research, Peking University Third HospitalCenter of Basic Medical Research, Peking University Third HospitalAbstract 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.https://doi.org/10.1186/s13104-025-07301-4Multiplexed immunohistochemistryAntibody strippingTyramide signal amplificationHybridization oven-based antibody removing (HO-AR) |
| spellingShingle | Xiaotong Yu Yan Song Li Chen Lixiang Xue Jianling Yang Chen Huang Optimization of a thermochemical antibody stripping method for enhanced tyramide signal amplification-based opal multiplex immunohistochemistry in fragile tissues BMC Research Notes Multiplexed immunohistochemistry Antibody stripping Tyramide signal amplification Hybridization oven-based antibody removing (HO-AR) |
| title | Optimization of a thermochemical antibody stripping method for enhanced tyramide signal amplification-based opal multiplex immunohistochemistry in fragile tissues |
| title_full | Optimization of a thermochemical antibody stripping method for enhanced tyramide signal amplification-based opal multiplex immunohistochemistry in fragile tissues |
| title_fullStr | Optimization of a thermochemical antibody stripping method for enhanced tyramide signal amplification-based opal multiplex immunohistochemistry in fragile tissues |
| title_full_unstemmed | Optimization of a thermochemical antibody stripping method for enhanced tyramide signal amplification-based opal multiplex immunohistochemistry in fragile tissues |
| title_short | Optimization of a thermochemical antibody stripping method for enhanced tyramide signal amplification-based opal multiplex immunohistochemistry in fragile tissues |
| title_sort | optimization of a thermochemical antibody stripping method for enhanced tyramide signal amplification based opal multiplex immunohistochemistry in fragile tissues |
| topic | Multiplexed immunohistochemistry Antibody stripping Tyramide signal amplification Hybridization oven-based antibody removing (HO-AR) |
| url | https://doi.org/10.1186/s13104-025-07301-4 |
| work_keys_str_mv | AT xiaotongyu optimizationofathermochemicalantibodystrippingmethodforenhancedtyramidesignalamplificationbasedopalmultipleximmunohistochemistryinfragiletissues AT yansong optimizationofathermochemicalantibodystrippingmethodforenhancedtyramidesignalamplificationbasedopalmultipleximmunohistochemistryinfragiletissues AT lichen optimizationofathermochemicalantibodystrippingmethodforenhancedtyramidesignalamplificationbasedopalmultipleximmunohistochemistryinfragiletissues AT lixiangxue optimizationofathermochemicalantibodystrippingmethodforenhancedtyramidesignalamplificationbasedopalmultipleximmunohistochemistryinfragiletissues AT jianlingyang optimizationofathermochemicalantibodystrippingmethodforenhancedtyramidesignalamplificationbasedopalmultipleximmunohistochemistryinfragiletissues AT chenhuang optimizationofathermochemicalantibodystrippingmethodforenhancedtyramidesignalamplificationbasedopalmultipleximmunohistochemistryinfragiletissues |