Universal, Rapid, and Cleavable Labeling of Antibodies by Fluorophores and DNA Oligonucleotides for Multiplex Immunostaining and Spatial Proteomics Through MIST Linker.

Direct antibody labeling is essential for immunoassays, multiplexed imaging, and biosensing; however, current methods are often time-consuming, restricted by antibody source, risk compromising protein performance, or vary with multiple steps. We introduce multiplex in situ tagging (MIST) Linker, a rapid and Fc-site-specific labeling tool that conjugates fluorophores or DNA oligonucleotides to antibodies from diverse commercial sources in as fast as 10 min using minimal starting material. MIST Linker achieves >90% cleavage upon UV exposure, facilitating rapid cyclic imaging on a single specimen. Validated across multiple species and sources, the platform outperforms conventional two-step immunofluorescence and immunohistochemistry in various tissues and cell lines. By enabling the rapid, cost-effective customization of antibody panels, MIST Linker significantly lowers the barrier to accessing antibody-DNA conjugates for spatial biology. When integrated with the spatial MIST platform and MIST-Explorer, it enables high-plex, single-cell spatial proteomics at high signal-to-noise ratios in human clinical biopsies, mouse specimens and cell lines. This toolkit provides an efficient, accessible solution for high-resolution spatial mapping, allowing for the in-depth analysis of cell subpopulations, biomarker distributions, and signaling events in complex biological specimens. Thus, MIST Linker offers a versatile, accessible, and scalable solution for antibody-labeling-based research and clinical diagnosis.