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This has the potential to maintain morphology for many decades, but has been found to cause progressive loss of antigenicity over time for some biomolecules, motivating interest in alternative long-term preservation strategies, such as cryopreservation. While cryoprotection and subzero storage has been successfully used for brain tissue sections or blocks, methods for preserving whole brains using this approach have not been widely characterized. Here we present a protocol for preserving fixed whole brains using graded immersion cryoprotection followed by subzero temperature storage. We refer to this general strategy \u2013 aldehyde fixation followed by cryoprotectant loading and subzero storage \u2013 as aldehyde-based cryopreservation (ABC). Our method uses a gradual ramp-up of the osmotic concentration of cryoprotectants, leading to a final solution containing 50% (v\/v) ethylene glycol and 30% (w\/v) sucrose in fixative. We used CT imaging to track cryoprotectant penetration, finding that approximately 9 months is required for the CT signal to stabilize throughout whole human brains. In our initial validation experiment, insufficient equilibration time prior to freezing led to ice crystal artifacts in the white matter. After refining the protocol to allow adequate diffusion time, light and electron microscopy showed preserved cellular architecture and ultrastructure. Our approach may be valuable for laboratories seeking a method for long-term subzero storage of fixed whole brain specimens.<\/jats:p>","DOI":"10.1371\/journal.pone.0344932","type":"journal-article","created":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T17:28:46Z","timestamp":1787592526000},"page":"e0344932","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["Cryopreservation of aldehyde-fixed whole brains"],"prefix":"10.1371","volume":"21","author":[{"given":"Macy","family":"Garrood","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alicia","family":"Keberle","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andria","family":"Slaughter","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Allison","family":"Sowa","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Emma L.","family":"Thorn","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Claudia","family":"De Sanctis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kurt","family":"Farrell","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John F.","family":"Crary","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7462-4340","authenticated-orcid":true,"given":"Andrew T.","family":"McKenzie","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,8,24]]},"reference":[{"key":"pone.0344932.ref001","first-page":"10","article-title":"Fluid preservation in brain banking: a review","volume":"5","author":"AT McKenzie","year":"2024","journal-title":"Free Neuropathol"},{"key":"pone.0344932.ref002","first-page":"35","article-title":"Cryopreservation of brain cell structure: a review","volume":"5","author":"AT McKenzie","year":"2024","journal-title":"Free Neuropathol"},{"key":"pone.0344932.ref003","doi-asserted-by":"crossref","first-page":"541","DOI":"10.1007\/BF00236191","article-title":"An improved HRP method for the study of central nervous connections","volume":"29","author":"JS de Olmos","year":"1977","journal-title":"Exp Brain Res"},{"issue":"1","key":"pone.0344932.ref004","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1016\/0196-9781(86)90076-8","article-title":"Use of cryoprotectant to maintain long-term peptide immunoreactivity and tissue morphology","volume":"7","author":"RE Watson","year":"1986","journal-title":"Peptides"},{"issue":"2","key":"pone.0344932.ref005","doi-asserted-by":"crossref","first-page":"171","DOI":"10.1002\/cne.903550203","article-title":"The human entorhinal cortex: a cytoarchitectonic analysis","volume":"355","author":"R Insausti","year":"1995","journal-title":"J Comp Neurol"},{"issue":"3","key":"pone.0344932.ref006","doi-asserted-by":"crossref","first-page":"28","DOI":"10.1017\/S155192950006106X","article-title":"Cryoprotectant tissue storage solutions: stability at lower temperatures, longer storage times, more versatile usage","volume":"8","author":"DM Simmons","year":"2000","journal-title":"Microscopy Today"},{"issue":"6","key":"pone.0344932.ref007","doi-asserted-by":"crossref","first-page":"2719","DOI":"10.1038\/nprot.2006.354","article-title":"Immunohistochemical staining of post-mortem adult human brain sections","volume":"1","author":"HJ Waldvogel","year":"2006","journal-title":"Nat Protoc"},{"issue":"3","key":"pone.0344932.ref008","first-page":"425","article-title":"Just cool it! 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