RT Journal Article SR Electronic T1 Highly multiplexed in situ protein imaging with signal amplification by Immuno-SABER JF bioRxiv FD Cold Spring Harbor Laboratory SP 507566 DO 10.1101/507566 A1 Sinem K. Saka A1 Yu Wang A1 Jocelyn Y. Kishi A1 Allen Zhu A1 Yitian Zeng A1 Wenxin Xie A1 Koray Kirli A1 Clarence Yapp A1 Marcelo Cicconet A1 Brian J. Beliveau A1 Sylvain W. Lapan A1 Siyuan Yin A1 Millicent Lin A1 Edward S. Boyden A1 Pascal S. Kaeser A1 German Pihan A1 George M. Church A1 Peng Yin YR 2018 UL http://biorxiv.org/content/early/2018/12/28/507566.abstract AB Probing the molecular organization of tissues requires in situ analysis by microscopy. However current limitations in multiplexing, sensitivity, and throughput collectively constitute a major barrier for comprehensive single-cell profiling of proteins. Here, we report Immunostaining with Signal Amplification By Exchange Reaction (Immuno-SABER), a rapid, highly multiplexed signal amplification method that simultaneously tackles these key challenges. Immuno-SABER utilizes DNA-barcoded antibodies and provides a method for highly multiplexed signal amplification via modular orthogonal DNA concatemers generated by Primer Exchange Reaction. This approach offers the capability to preprogram and control the amplification level independently for multiple targets without in situ enzymatic reactions, and the intrinsic scalability to rapidly amplify and image a large number of protein targets. We validated our approach in diverse sample types including cultured cells, cryosections, FFPE sections, and whole mount tissues. We demonstrated independently tunable 5-180-fold amplification for multiple targets, covering the full signal range conventionally achieved by secondary antibodies to tyramide signal amplification, as well as simultaneous signal amplification for 10 different proteins using standard equipment and workflow. We further combined Immuno-SABER with Expansion Microscopy to enable rapid and highly multiplexed super-resolution tissue imaging. Overall, Immuno-SABER presents an effective and accessible platform for rapid, multiplexed imaging of proteins across scales with high sensitivity.