Abunmix enables the simple and robust multiplexed immunofluorescence imaging

Over the past 10 years, spatial proteomics has attracted significant attention, not only in neuroscience and cancer research but also in developmental biology, drug delivery studies, and organoid research. To meet this growing demand, various multiplexed immunofluorescence imaging techniques have been developed. However, despite these advances, one challenge remains for these techniques to be more widely used in typical laboratories: the limited availability of host species of antibodies. In this work, a technique termed Abunmix, enabling indirect staining using primary antibodies produced from the same host species, is demonstrated. In Abunmix, primary and secondary antibodies conjugated to different fluorophores are applied to specimens sequentially. After staining, multichannel images are acquired. Since primary antibodies from the same host are used, the resulting images contain mixed signals from multiple proteins. Then, signal‐unmixing algorithms are applied to separate mixed signals into the signals of each protein. Using this approach, multiplexed imaging via indirect staining of multiple same‐host primary antibodies is achieved using typical primary and secondary antibodies, without any special chemicals or instruments. Given its minimal experimental requirements, Abunmix will accelerate the adoption of multiplexed immunofluorescence imaging, advancing research and diagnostics across diverse biological fields.