Fig. 1: Schematic of multiExR procedure.

a Expansion revealing (ExR), a technology for decrowding of proteins through isotropic protein separation. ai Coronal section of mouse brain before staining or expansion. aii Anchoring and first gelation step. The specimen is embedded in a swellable hydrogel (gray wavy lines), mechanically softened via detergent and heat treatment, and expanded in water. aiii Re-embedding and second swellable gel formation. The fully expanded first gel is re-embedded in a charge-neutral gel (not shown), followed by the formation of a second swellable hydrogel (light gray wavy lines). aiv Final up to 20x expansion with the addition of water, followed by a recommended re-embedding step to preserve gel strength for multi-round imaging (blue wavy lines). av, Post-expansion primary antibody staining (Y-shaped proteins). avi Post-expansion staining with fluorescent secondary antibodies to visualize decrowded biomolecules. b Multiplexed ExR procedure. bi Free-floating gels are stained with conventional primary and secondary antibodies, and the images are collected. bii After imaging, primary and secondary antibodies are stripped using detergent and heat-based denaturation while endogenous proteins are preserved by physical anchoring in hydrogel networks. biii Gels are re-incubated with a new round of primary and secondary antibodies, and the same field of view is imaged again. biv A 3 or 4-channel z-stack is obtained on a confocal microscope. One or more of the four channels serves as the reference channel. After imaging, the antibody stripping and staining processes are repeated for up to 10 rounds. c Registration of multi-round images using the reference channel. The multi-round images are registered using one or a combination of the methods (i-a and i-c, or i-b and i-c) in this toolbox (see Supplementary Fig. 1 and “Methods” section for more details). i-a a feature-based affine registration algorithm^8,9. i-b an intensity-based affine registration algorithm¹⁰ iteratively refining the estimation from the coarse scale of the image pairs to the fine scale. i-c, a point-based registration algorithm¹¹, designed specifically to further align fine structures. cii Registered multiExR images are obtained after applying calculated warps to all channels from later rounds, creating multi-channel image volumes. Schematic created with BioRender.com. Bolded, green text highlights technical innovations of the multiExR procedure.