Proteins work together in nanostructures in many physiological contexts and disease states. We recently developed expansion revealing (ExR), which expands proteins away from each other, in order to support better labeling with antibody tags and nanoscale imaging on conventional microscopes. Here, we report multiplexed expansion revealing (multiExR), which enables high-fidelity antibody visualization of >20 proteins in the same specimen, over serial rounds of staining and imaging. Across all datasets examined, multiExR exhibits a median round-to-round registration error of 39 nm, with a median registration error of 25 nm when the most stringent form of the protocol is used. We precisely map 23 proteins in the brain of 5xFAD Alzheimer’s model mice, and find reductions in synaptic protein cluster volume, and co-localization of specific AMPA receptor subunits with amyloid-beta nanoclusters. We visualize 20 synaptic proteins in specimens of mouse primary somatosensory cortex. multiExR may be of broad use in analyzing how different kinds of protein are organized amidst normal and pathological processes in biology.
