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Updated: Nov 10, 2025
Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging
Published on: April 8, 2016
[Practical Basis of Immunohistochemical Research and Multiplexed Immunohistochemistry]
1Dept. of Pathology, Institute of Medical Science, Tokyo Medical University.
Insights
Immunohistochemistry (IHC) visualizes antigen distribution in tissues. Multiplex IHC (mIHC) advances this by enabling mathematical analysis of cell coordinates and antigen expression for enhanced research reproducibility.
Area of Science:
- Cellular and Molecular Biology
- Histopathology
- Biomedical Imaging
Background:
- Immunohistochemistry (IHC) is crucial for visualizing in situ antigen distribution in research and diagnostics.
- Traditional IHC image interpretation can lack reproducibility compared to methods like flow cytometry.
- Multiplex IHC (mIHC) has emerged, offering advanced capabilities for tissue analysis.
Purpose of the Study:
- To reconfirm fundamental principles for performing accurate IHC.
- To present methodologies for multiplexed IHC, including mIHC.
- To discuss the features, advantages, and unresolved challenges of mIHC.
Main Methods:
- Review of basic IHC principles and sample preparation.
- Description of various methodological approaches for multiplexed IHC (mIHC).
- Discussion of data output as numerical matrices for mathematical analysis.
Main Results:
- mIHC transforms morphological data (cell coordinates, antigen levels) into quantifiable matrices.
- This facilitates mathematical analysis of tissue morphology, improving objectivity.
- The study outlines current mIHC techniques and their applications.
Conclusions:
- Proper IHC requires understanding principles, sample prep, antibody selection, and methods.
- mIHC significantly enhances tissue analysis by enabling quantitative, reproducible insights.
- Further research is needed to address the remaining challenges in mIHC technology.
Abstract:
Immunohistochemistry(IHC)is a central tool of modern morphology. IHC is widely used from basic research to diagnostic practice utilizing its advantage in visualizing ability of in situ distribution of target antigens. In order to perform proper immunohistochemical analyses, it is necessary to fully understand the principle of IHC to prepare appropriate samples, to select suitable antibodies, and to apply reasonable method. The final output of IHC and conventional multi-labeled IHC, are given as histological images. Interpretation of those images is entrusted to the researcher. Therefore, reproducibility and verifiability of those subjective explanation of IHC images are occasionally insufficient as compared to other experimental methods such as(multi-colored)flow cytometry. In recent years, extremely multiplexed IHC method(multiplex immunohistochemistry: mIHC)by various methodological approach have been developed and put into practical use. By mIHC, morphological factors, namely coordinate of cells and expression level of target antigens, are established as matrices of numeral values, and mathematical analyses of tissue morphology is now practical. In this essay, basic matters to keep in mind at performing proper IHC are reconfirmed, and present a methodology of multiplexed IHC including mIHC. In addition to their features and advantages, unsolved issues of mIHC are also taken up.
