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. 2022 Nov 30;13:7242. doi: 10.1038/s41467-022-34824-2

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© The Author(s) 2022

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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — A Chemically fixed tissue samples, which can be immunostained beforehand, are treated with the chemical anchor, embedded into the hydrogel, and mechanically homogenized by mild denaturation. The Coomassie brilliant blue (CBB) hydrogel embedded samples are expanded and imaged. After imaging, expanded samples are microdissected and excised pieces of the tissue-hydrogel composite are processed by in-gel digestion to recover peptides for LC-MS/MS analysis (LEF linear expansion factor, VOL volumetric expansion factor). Created with Biorender.com. B Timeline of ProteomEx indicating total duration and hands-on time of each step (total duration/hands-on time). C Representative brightfield images of mouse brain tissue section before expansion and (D) after Coomassie staining and expansion (E) showing automatically detected and annotated brain regions (LEF = 5.5-fold; n = 20 brain slices from 16 mice).