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. 2024 Apr 24;21(9):1702–1707. doi: 10.1038/s41592-024-02242-5

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 2 — 2-plex Exchange-PAINT image of transiently transfected BSC1 cells expressing the monomeric membrane receptor CD86. For characterization of intracellular tag binders targeting mEGFP, ALFA-tag, mCherry, TagRFP, mNeonGreen, mEOS2 or SPOT-tag, the intracellular tag of interest was inserted at the CD86 C-terminus and the reference tag (for example ALFA-tag, mEGFP) was inserted at the CD86 N-terminus on the extracellular side of the cell surface. To notably boost the labeling efficiency against a single target protein, For mEGFP, nanobody clones were combined as well as ALFA-tag and mEGFP were concatenated at theCD86 C-terminus to boost labeling efficiency. For concatenated tag, anti-CD86 antibody served as a reference. A fluorescent tag (for example mEGFP, mCherry) served as an indicator for construct expression. Zoom-ins depict super-resolution representation of target binder (cyan) colocalizing with respective reference binder signal (magenta).