Ruthenium Counterstaining for Imaging Mass Cytometry

Catena, R., Montuenga, L.M., Bodenmiller, B.

Imaging mass cytometry is a novel imaging modality that enables simultaneous antibody-based detection of more than 40 epitopes and molecules in tissue sections at subcellular resolution using isotopically pure metal tags. Essential for any imaging approach where antigen detection is performed is the so-called counterstaining that reveals the overall structure of the tissue. Counterstaining is necessary since antigens of interest are often present only in a small subset of cells while the rest of tissue structures are not visible. Since most biological tissues are nearly transparent or non-fluorescent, chromogenic reagents such as haematoxylin (for immunohistochemistry) or fluorescent dyes such as DAPI (that 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.


Catena, R., Montuenga, L.M., Bodenmiller, B. "Ruthenium Counterstaining for Imaging Mass Cytometry" Journal of Pathology (2018): 479–84