Technological breakthroughs have fundamentally changed our understanding on the complexity of tissue organization, both in healthy and diseased conditions. Characterizing the immune cell composition in relation to spatial distribution and histological changes may provide important diagnostic and therapeutic information. Immunohistochemistry remains a method of choice for these purposes, with crucial implications in clinics or in medical research. Nowadays, the widespread use of fluorophore-conjugated antibodies enables simultaneous visualization of an increasing number of proteins on a single tissue slice, with unprecedented resolution. However, advanced methods usually require modern and specific equipment, a significant amount of time for assay optimization, and highly specialized skills. This work reports on the use of a multiplex immunostaining method based on sequential immunostaining and antibody stripping, combined with digital image processing and analysis. Our aim is to provide the medical and research communities with a simple, cost-effective workflow to encompass some current limitations of multiplex immunohistochemistry.

Technological breakthroughs have fundamentally changed our understanding on the complexity of the tumor microenvironment at the single-cell level. Characterizing the immune cell composition in relation to spatial distribution and histological changes may provide important diagnostic and therapeutic information. Immunostaining on formalin-fixed paraffin-embedded (FFPE) tissue samples represents a widespread and simple procedure, allowing the visualization of cellular distribution and processes, on preserved tissue structure. Recent advances in microscopy and molecular biology have made multiplexing accessible, yet technically challenging. We herein describe a novel, simple and cost-effective method for a reproducible and highly flexible multiplex immunostaining on archived FFPE tissue samples, which we optimized for solid organs (e.g., liver, intestine, lung, kidney) from mice and humans. Our protocol requires limited specific equipment and reagents, making multiplexing (>12 antibodies) immediately implementable to any histology laboratory routinely performing immunostaining. Using this method on single sections and combining it with automated whole-slide image analysis, we characterize the hepatic immune microenvironment in preclinical mouse models of liver fibrosis, steatohepatitis and hepatocellular carcinoma (HCC) and on human-patient samples with chronic liver diseases. The data provide useful insights into tissue organization and immune–parenchymal cell-to-cell interactions. It also highlights the profound macrophage heterogeneity in liver across premalignant conditions and HCC.