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bioRxiv · 10.64898/2026.09.11.750056

Spatial organization and mitigation of autofluorescence in multiplexed spatial proteomics of aged fresh-frozen human brain

Sutevski, I.·Guo, L.·Branzell, N.·Andren, P. E.·Svenningsson, P.·Ayoglu, B.
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Abstract

Multiplexed imaging technologies are transforming the study of human tissue biology, but their application to the aged brain is hindered by autofluorescence, particularly in fresh-frozen specimens. Here, we characterized autofluorescence across four brain regions from 21 donors and found broad spectral emission, regional and gray-white matter differences, and an association with donor age. Photobleaching conditions adopted from formalin-fixed paraffin-embedded tissue caused marked region- and compartment-dependent damage in fresh-frozen sections. We therefore developed a Tris-EDTA-supplemented photobleaching workflow that reduced autofluorescence by 58-70% while preserving tissue architecture and cellular content. We established a custom 28-plex DNA-barcoded antibody panel targeting neuronal, glial, immune, and vascular markers, providing a resource for fresh-frozen human brain. Integration of the optimized photobleaching workflow with this panel enabled spatial proteomic analysis across fresh-frozen brain regions. By co-registering pre-photobleaching autofluorescence with multiplexed protein maps, we further established a cellular-resolution framework for spatial characterization of autofluorescence. This revealed region-dependent protein marker relationships and preferential enrichment of autofluorescent particles near nuclei and within microglial and CD68-positive regions. Together, this work establishes a practical workflow for multiplexed spatial proteomics in fresh-frozen brain and characterizes autofluorescence as both a technical confound and a spatially structured feature of the aged human brain.

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Sutevski, I., Guo, L., Branzell, N., Andren, P. E., Svenningsson, P., Ayoglu, B.. 2026-09-18. Spatial organization and mitigation of autofluorescence in multiplexed spatial proteomics of aged fresh-frozen human brain. https://doi.org/10.64898/2026.09.11.750056

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