AbstractImaging mass cytometry is a novel imaging modality that enables simultaneous antibody‐based detection of >40 epitopes and molecules in tissue sections at subcellular resolution by the use of isotopically pure metal tags. Essential for any imaging approach in which antigen detection is performed is counterstaining, which reveals the overall structure of the tissue. Counterstaining is necessary because antigens of interest are often present in only a small subset of cells, and the rest of the tissue structures are not visible. As most biological tissues are nearly transparent or non‐fluorescent, chromogenic reagents such as haematoxylin (for immunohistochemistry) or fluorescent dyes such as 4′,6‐diamidino‐2‐phenylindole (which stains nuclei for epifluorescence and confocal microscopy) are utilized. Here, we describe a metal‐based counterstain for imaging mass cytometry based on simple oxidation and subsequent covalent binding of the tissue components to ruthenium tetroxide (RuO4). RuO4 counterstaining reveals general tissue structure both in areas with high cell content and in stromal areas with low cellularity and fibrous or hyaline material in a manner analogous to haematoxylin in immunohistochemical counterstaining or eosin or other anionic dyes in conventional histology. Our new counterstain approach is applicable to any metal‐based imaging technique, and will facilitate the adaptation of imaging mass cytometry for routine applications in clinical and research laboratories. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.