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Automated quantitative high‑throughput multiplex immunofuorescence pipeline to evaluate OXPHOS defects in formalin‑fxed human prostate tissue

Lookup NU author(s): Dr Ashwin Sachdeva, Dr Christopher Carey, Dr Amy VincentORCiD, Dr Laura Greaves, Professor Rakesh Heer, Emeritus Professor Doug Turnbull



This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


Advances in multiplex immunofuorescence (mIF) and digital image analysis has enabled simultaneous assessment of protein defects in electron transport chain components. However, current manual methodology is time consuming and labour intensive. Therefore, we developed an automated highthroughput mIF workfow for quantitative single-cell level assessment of formalin fxed parafn embedded tissue (FFPE), leveraging tyramide signal amplifcation on a Ventana Ultra platform coupled with automated multispectral imaging on a Vectra 3 platform. Utilising this protocol, we assessed the mitochondrial oxidative phosphorylation (OXPHOS) protein alterations in a cohort of benign and malignant prostate samples. Mitochondrial OXPHOS plays a critical role in cell metabolism, and OXPHOS perturbation is implicated in carcinogenesis. Marked inter-patient, intra-patient and spatial cellular heterogeneity in OXPHOS protein abundance was observed. We noted frequent Complex IV loss in benign prostate tissue and Complex I loss in age matched prostate cancer tissues. Malignant regions within prostate cancer samples more frequently contained cells with low Complex I & IV and high mitochondrial mass in comparison to benign–adjacent regions. This methodology can now be applied more widely to study the frequency and distribution of OXPHOS alterations in formalin-fxed tissues, and their impact on long-term clinical outcomes.

Publication metadata

Author(s): Sachdeva A, Hart CA, Carey CD, Vincent AE, Greaves LC, Heer R, Oliveira P, Brown MD, Clarke NW, Turnbull DM

Publication type: Article

Publication status: Published

Journal: Scientific Reports

Year: 2022

Volume: 12

Pages: 6660

Online publication date: 22/04/2022

Acceptance date: 01/04/2022

Date deposited: 03/05/2022

ISSN (electronic): 2045-2322

Publisher: Nature


DOI: 10.1038/s41598-022-10588-z

PubMed id: 35459777


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