Related Experiment Video
Updated: May 4, 2026
High-Throughput Automated Multiplex Immunofluorescence Assays for Translational Research
Published on: June 10, 2025
Evaluating real-time immunohistochemistry on multiple tissue samples, multiple targets and multiple antibody labeling
Louise Dubois, Karl Andersson, Anna Asplund
1Department of Radiology, Oncology, and Radiation Sciences; Biomedical Radiation Sciences, Rudbeck Laboratory, Uppsala University, 75185 Uppsala, Sweden. hanna.bjorkelund@bms.uu.se.
Insights
Real-time immunohistochemistry (RT-IHC) offers a standardized, quantitative method for analyzing protein expression in tissues. This technique tracks antibody-antigen interactions over time, improving assay optimization and sensitivity in pathology.
Area of Science:
- Biotechnology
- Molecular Biology
- Pathology
Background:
- Immunohistochemistry (IHC) is a standard pathology technique for protein expression analysis.
- Current IHC methods involve subjective interpretation and lack standardization, leading to variability.
- Real-time immunohistochemistry (RT-IHC) offers a novel, operator-independent approach for time-resolved antibody-target protein interactions.
Purpose of the Study:
- To evaluate the technical aspects and general applicability of RT-IHC.
- To determine the quantitative potential of RT-IHC for protein analysis.
- To understand antibody-antigen kinetics for optimizing conventional IHC protocols.
Main Methods:
- Applied three different antibodies (fluorescent or radioactive labels) to nine human and mouse tissue samples.
- Utilized RT-IHC to capture time-resolved binding curves of antibody-antigen interactions.
- Assessed the impact of tissue size, thickness, and section position on signal magnitude and binding kinetics.
Main Results:
- RT-IHC demonstrated general applicability across various antibodies and tissue types.
- Most antibody-antigen interactions did not reach equilibrium within 3 hours, indicating potential under-optimization in standard IHC.
- Binding curve curvature, reflecting interaction kinetics, was independent of tissue size, thickness, and position, suggesting a robust quantitative parameter.
Conclusions:
- RT-IHC is a versatile and generalizable method for evaluating antibody-antigen interactions.
- Analyzing binding kinetics over time provides insights for optimizing conventional IHC assays.
- RT-IHC has the potential to improve sensitivity and standardization in protein expression analysis.
Background:
Immunohistochemistry (IHC) is a well-established method for the analysis of protein expression in tissue specimens and constitutes one of the most common methods performed in pathology laboratories worldwide. However, IHC is a multi-layered method based on subjective estimations and differences in staining and interpretation has been observed between facilities, suggesting that the analysis of proteins on tissue would benefit from protocol optimization and standardization. Here we describe how the emerging and operator independent tool of real-time immunohistochemistry (RT-IHC) reveals a time resolved description of antibody interacting with target protein in formalin fixed paraffin embedded tissue. The aim was to understand the technical aspects of RT-IHC, regarding generalization of the concept and to what extent it can be considered a quantitative method.
Results:
Three different antibodies labeled with fluorescent or radioactive labels were applied on nine different tissue samples from either human or mouse, and the results for all RT-IHC analyses distinctly show that the method is generally applicable. The collected binding curves showed that the majority of the antibody-antigen interactions did not reach equilibrium within 3 hours, suggesting that standardized protocols for immunohistochemistry are sometimes inadequately optimized. The impact of tissue size and thickness as well as the position of the section on the glass petri dish was assessed in order for practical details to be further elucidated for this emerging technique. Size and location was found to affect signal magnitude to a larger extent than thickness, but the signal from all measurements were still sufficient to trace the curvature. The curvature, representing the kinetics of the interaction, was independent of thickness, size and position and may be a promising parameter for the evaluation of e.g. biopsy sections of different sizes.
Conclusions:
It was found that RT-IHC can be used for the evaluation of a number of different antibodies and tissue types, rendering it a general method. We believe that by following interactions over time during the development of conventional IHC assays, it becomes possible to better understand the different processes applied in conventional IHC, leading to optimized assay protocols with improved sensitivity.
