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The Opal methodology allows up to eight biomarkers to be measured concomitantly without cross\u2010reactivity, permitting identification of different cell populations within the tumour microenvironment. In this study, we aimed to validate a multiplex immunofluorescence workflow in two complementary multiplex panels and evaluate the tumour immune microenvironment in colorectal cancer (CRC) formalin\u2010fixed paraffin\u2010embedded tissue. We stained CRC and tonsil samples using Opal multiplex immunofluorescence on a Leica BOND RX immunostainer. We then acquired images on an Akoya Vectra Polaris and performed multispectral unmixing using <jats:sc>inform<\/jats:sc>. Antibody panels were validated on tissue microarray sections containing cores from six normal tissue types, using <jats:sc>qupath<\/jats:sc> for image analysis. Comparisons between chromogenic immunohistochemistry and multiplex immunofluorescence on consecutive sections from the same tissue microarray showed significant correlation (<jats:italic>r<\/jats:italic><jats:sub>s<\/jats:sub>\u00a0&gt;\u00a00.9, <jats:italic>P<\/jats:italic>\u2010value\u00a0&lt;\u00a00.0001), validating both panels. We identified many factors that influenced the quality of the acquired fluorescent images, including biomarker co\u2010expression, staining order, Opal\u2010antibody pairing, sample thickness, multispectral unmixing and biomarker detection order during image analysis. Overall, we report the optimisation and validation of a multiplex immunofluorescence process, from staining to image analysis, ensuring assay robustness. Our multiplex immunofluorescence protocols permit the accurate detection of multiple immune markers in various tissue types, using a workflow that enables rapid processing of samples, above and beyond previous workflows.<\/jats:p>","DOI":"10.1002\/1878-0261.12764","type":"journal-article","created":{"date-parts":[[2020,7,16]],"date-time":"2020-07-16T08:41:25Z","timestamp":1594888885000},"page":"2384-2402","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":112,"title":["A robust multiplex immunofluorescence and digital pathology workflow for the characterisation of the tumour immune microenvironment"],"prefix":"10.1002","volume":"14","author":[{"given":"Am\u00e9lie","family":"Viratham Pulsawatdi","sequence":"first","affiliation":[{"name":"Patrick G Johnston Centre for Cancer Research Queen's University Belfast Belfast UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephanie 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