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bioRxiv · 10.64898/2026.09.18.751896

Ultrastructural comparison of fixation and cryopreservation methods for brain preservation

German, A.·Fluegel-Koch, C.·Paulsen, F.·Winkler, J.·McKenzie, A. T.
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Abstract

Maximizing the morphological and molecular fidelity of preserved mammalian brain tissue is essential for basic neuroscience and brain banking, where tissue quality determines the reliability of downstream analyses. Aldehyde fixation and cryogenic storage are the most powerful preservation techniques available. However, how to best combine them is incompletely characterized. Here, we tested twelve different approaches to preserve the murine brain, including standard aldehyde perfusion fixation, variants of aldehyde-stabilized cryopreservation (ASC) using ethylene glycol (EG), aldehyde fixation followed by unprotected freezing, and several cryopreservation protocols without preceding aldehyde fixation, such as interleaved equilibration with vitrification solution. Preservation quality was assessed using light microscopy, transmission electron microscopy, and patch-level feature analysis with the DINOv2 vision foundation model. We found that ASC with sodium dodecyl sulfate (SDS)-mediated blood-brain barrier permeabilization preserved nuclear morphology, myelin periodicity, and neuropil texture comparable to standard aldehyde perfusion fixation, providing the first independent replication of ASC. Omitting SDS caused severe parenchymal dehydration, due to a mismatch between water and cryoprotectant transport. Aldehyde fixation followed by unprotected slow freezing confined ice damage primarily to perivascular zones, while fast freezing led to intranuclear clefts and cavities. Among non-fixation protocols, interleaved equilibration with vitrification solution preserved myelinated axon profiles and chromatin patterns, but induced perivascular edema. High concentration glycerol perfusion caused significant osmotic dehydration. Straight freezing of unfixed tissue without cryoprotectant was followed by membrane disruption, but resulted in the visualization of more structure than expected, likely as a result of structural restitution upon thawing. Taken together, our results provide a framework for matching preservation strategies to the needs of different types of brain banking and neuroscience research.

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German, A., Fluegel-Koch, C., Paulsen, F., Winkler, J., McKenzie, A. T.. 2026-09-24. Ultrastructural comparison of fixation and cryopreservation methods for brain preservation. https://doi.org/10.64898/2026.09.18.751896

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