Background
Multiplexed immunohistochemistry (IHC) has the potential to improve conventional IHC staining allowing for analysis of multiple cell phenotypes while maintaining spatial context. Automated multispectral image analysis and computer-based cell recognition make the process more attainable, but stringent validation of multiplex IHC is still required. Pertinently, multiplex IHC allows for the characterisation and enumeration of immune cell densities in the tumour micro-environment, of particular importance for analysis of tumour-infiltrating immune cells which require multiple co-localised markers for their identification. However, issues of antibody blocking, cross-reactivity and masking have caused concern that the results of multiplex staining may not accurately reflect those of single-plex staining. The Opal workflow from PerkinElmer uses a heat-induced epitope retrieval step between each antibody detected which aims to obviate these potential problems and enable single-species antibody use. In this study we systematically validated multiplex staining for a range of immune cell markers against single-plex staining for each marker to determine the accuracy of the multiplex method.