Background: By simultaneously staining multiple immunomarkers on a single tissue section, multiplexed immunohistochemistry (mIHC) enhances the amount of information that can be observed in a single tissue section and thus can be a powerful tool to visualise cellular interactions directly in the tumour microenvironment. Performing mIHC remains technically and practically challenging, and this technique has many limitations if not properly validated. However, with proper validation, heterogeneity between histopathological images can be avoided.
Aims: This review aimed to summarize the currently used methods and to propose a standardised method for effective mIHC.
Materials and methods: An extensive literature review was conducted to identify different methods currently in use for mIHC.
Results: Guidelines for antibody selection, panel design, antibody validation and analytical strategies are given. The advantages and disadvantages of each method are discussed.
Conclusion: This review summarizes widely used pathology imaging software and discusses the potential for automation of pathology image analysis so that mIHC technology can be a truly powerful tool for research as well as clinical use.
Keywords: IHC; cancer; histopathology; mIHC; multiplex immunohistochemistry; tumour biomarkers.
© 2025 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.