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. 2021 Jun 2;8:674747. doi: 10.3389/fmolb.2021.674747

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Copyright © 2021 Hoyt.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Mechanism of tyramide signal amplification (TSA) staining. Opal dyes allow for the use of any standard unlabeled primary antibody, including multiple antibodies raised in the same species. After introduction of the primary antibody, the Opal polymer HRP is applied. The Opal system uses TSA to amplify IHC detection by covalently depositing multiple fluorophores near that targeted antigen. After labeling is complete, antibodies are removed in a manner that does not disrupt the Opal fluorescence signal, allowing for the next target to be detected without antibody cross-reactivity.