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Published November 15, 2021 | Supplemental Material + Submitted + Published
Journal Article Open

Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization


RNA in situ hybridization based on the mechanism of the hybridization chain reaction (HCR) enables multiplexed, quantitative, high-resolution RNA imaging in highly autofluorescent samples, including whole-mount vertebrate embryos, thick brain slices and formalin-fixed paraffin-embedded tissue sections. Here, we extend the benefits of one-step, multiplexed, quantitative, isothermal, enzyme-free HCR signal amplification to immunohistochemistry, enabling accurate and precise protein relative quantitation with subcellular resolution in an anatomical context. Moreover, we provide a unified framework for simultaneous quantitative protein and RNA imaging with one-step HCR signal amplification performed for all target proteins and RNAs simultaneously.

Additional Information

© 2021. Published by The Company of Biologists Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. Received 30 May 2021; Accepted 12 October 2021. We thank F. Chen for discussions, M. E. Bronner for reading a draft of the manuscript and the following resources within the Beckman Institute at Caltech: A. Collazo of the Biological Imaging Facility for assistance with imaging, G. Shin of Molecular Technologies for providing HCR reagents and the Zebrafish Facility for providing zebrafish embryos. This work was funded by the National Institutes of Health (NIBIB R01EB006192, NIGMS R44GM140796 and NIGMS training grant GM008042 to S.J.S.), by the Defense Advanced Research Projects Agency (HR0011-17-2-0008; the findings are those of the authors and should not be interpreted as representing the official views or policies of the U.S. Government) and by the Beckman Institute, California Institute of Technology (Programmable Molecular Technology Center, PMTC). Open access funding provided by the California Institute of Technology. Deposited in PMC for immediate release. Author contributions: Conceptualization: N.A.P.; Methodology: M.S., M.C.L., S.J.S., N.H., H.M.T.C., N.A.P.; Validation: M.S., S.J.S., R.I.; Investigation: M.S., M.C.L., S.J.S., R.I., N.H.; Writing - original draft: N.A.P.; Writing - review & editing: M.S., M.C.L., S.J.S., R.I., N.H., H.M.T.C., N.A.P.; Visualization: M.S., S.J.S., R.I.; Supervision: M.C.L., H.M.T.C., N.A.P.; Project administration: H.M.T.C., N.A.P.; Funding acquisition: H.M.T.C., N.A.P. The authors declare competing financial interests in the form of patents, pending patent applications and the startup company Molecular Instruments.

Attached Files

Published - dev199847.pdf

Submitted - 2021.06.02.446311v1.full.pdf

Supplemental Material - dev199847supp.pdf


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August 20, 2023
December 22, 2023