{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,26]],"date-time":"2026-10-26T13:21:17Z","timestamp":1793020877160,"version":"4.5.0"},"reference-count":44,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2024,10,9]],"date-time":"2024-10-09T00:00:00Z","timestamp":1728432000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Neurosci."],"abstract":"<jats:p>Postmortem human brain tissue is a critical resource for studying neurodegenerative disease, providing critical insights into cellular morphology, pathology, and network connectivity. To improve standard microscopy and enable high-resolution, three-dimensional (3D) images of tissues at the subcellular level, tissue-clearing methods have been developed. These 3D images allow for the analysis of large regions of interest and can be used to study structural and spatial changes that occur during neurodegeneration. Additionally, 3D imaging facilitates the visualization of whole-cell morphology, especially in cells with long processes that would otherwise be truncated in single-plane images. Human brain tissue is especially challenging for tissue clearing due to the abundance of lipids in myelin and the need for optimal fixation and low postmortem intervals. Formaldehyde-based fixatives, commonly used in preserving tissue, hinder antibody binding by crosslinking important antibody epitopes, and fluorescent microscopy requires the incorporation of fluorescent labels through passive diffusion or electrophoresis. Recent studies have focused on optimally fixed human brain tissue with short postmortem intervals, limiting the general applicability of these methods. To address these challenges, we developed SHARD (SHIELD, antigen retrieval, and delipidation), a simple and widely applicable method for clearing and labeling human brain tissue, which can be applied to long-term banked human brain tissue preserved in formaldehyde. SHARD is a novel addition to the SHIELD tissue clarification method, combining antigen retrieval, tissue clearing, and staining of 200-\u03bcm sections from long-term banked human brain tissue. The SHARD method is effective for postmortem intervals (PMIs) ranging from 10 to 72\u2009h in multiple neurodegenerative diseases and control samples. In this study, we demonstrate that the SHARD method significantly enhances the immunostaining of glial fibrillary acidic protein (GFAP), an astrocytic cytoskeletal marker. Overall, the combination of antigen retrieval and tissue delipidation holds great potential for achieving detailed 3D immunostaining in long-term formaldehyde-fixed postmortem human brain tissue, opening new avenues for research and discovery.<\/jats:p>","DOI":"10.3389\/fnins.2024.1474617","type":"journal-article","created":{"date-parts":[[2024,10,9]],"date-time":"2024-10-09T01:10:26Z","timestamp":1728436226000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":7,"title":["SHARD: an improved method for staining and visualizing multiplex immunofluorescence in optically cleared postmortem human brain tissue"],"prefix":"10.3389","volume":"18","author":[{"given":"Grace A.","family":"Rosen","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel","family":"Kirsch","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Raymond","family":"Nicks","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hunter","family":"Kelley","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rebecca","family":"Mathias","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kerry A.","family":"Cormier","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Caroline A.","family":"Kubilus","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bryan","family":"Dec","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thor D.","family":"Stein","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Victor E.","family":"Alvarez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael L.","family":"Alosco","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ann C.","family":"McKee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bertrand R.","family":"Huber","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2024,10,9]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"e3378","DOI":"10.1371\/journal.pone.0003378","article-title":"A new antigen retrieval technique for human brain tissue","volume":"3","author":"Alel\u00fa-Paz","year":"2008","journal-title":"PLoS One"},{"key":"ref2","doi-asserted-by":"publisher","first-page":"193","DOI":"10.1186\/s13195-021-00928-y","article-title":"Structural MRI profiles and tau correlates of atrophy in autopsy-confirmed CTE","volume":"13","author":"Alosco","year":"2021","journal-title":"Alzheimers Res. 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Methods"},{"key":"ref10","doi-asserted-by":"publisher","first-page":"1983","DOI":"10.1038\/nprot.2012.119","article-title":"Three-dimensional imaging of solvent-cleared organs using 3DISCO","volume":"7","author":"Ert\u00fcrk","year":"2012","journal-title":"Nat. Protoc."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"163","DOI":"10.1016\/0165-0270(94)90208-9","article-title":"Effects of microwave pretreatment on immunocytochemical staining of vibratome sections and tissue blocks of human cerebral cortex stored in formaldehyde fixative for long periods","volume":"55","author":"Evers","year":"1994","journal-title":"J. Neurosci. Methods"},{"key":"ref12","doi-asserted-by":"publisher","first-page":"73","DOI":"10.1007\/s11920-017-0822-6","article-title":"VA\u2019s national PTSD brain Bank: a National Resource for research","volume":"19","author":"Friedman","year":"2017","journal-title":"Curr. Psychiatry Rep."},{"key":"ref13","doi-asserted-by":"publisher","first-page":"108","DOI":"10.1080\/09084282.2011.643947","article-title":"Diagnostic accuracy statistics for seven neuropsychological assessment battery (NAB) test variables in the diagnosis of Alzheimer\u2019s disease","volume":"19","author":"Gavett","year":"2012","journal-title":"Appl. Neuropsychol. Adult"},{"key":"ref14","doi-asserted-by":"publisher","first-page":"10880","DOI":"10.1038\/s41598-019-47336-9","article-title":"Scalable labeling for Cytoarchitectonic characterization of large optically cleared human neocortex samples","volume":"9","author":"Hildebrand","year":"2019","journal-title":"Sci. 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A."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"609","DOI":"10.1038\/s41592-020-0823-y","article-title":"Elasticizing tissues for reversible shape transformation and accelerated molecular labeling","volume":"17","author":"Ku","year":"2020","journal-title":"Nat. Methods"},{"key":"ref19","doi-asserted-by":"publisher","first-page":"1066","DOI":"10.1038\/s41467-018-03359-w","article-title":"Next generation histology methods for three-dimensional imaging of fresh and archival human brain tissues","volume":"9","author":"Lai","year":"2018","journal-title":"Nat. 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Protoc."},{"key":"ref23","doi-asserted-by":"publisher","first-page":"3506","DOI":"10.1038\/s41596-019-0240-9","article-title":"Advanced CUBIC tissue clearing for whole-organ cell profiling","volume":"14","author":"Matsumoto","year":"2019","journal-title":"Nat. Protoc."},{"key":"ref24","doi-asserted-by":"publisher","first-page":"239","DOI":"10.1038\/s41596-020-00414-z","article-title":"Antigen retrieval and clearing for whole-organ immunofluorescence by FLASH","volume":"16","author":"Messal","year":"2021","journal-title":"Nat. Protoc."},{"key":"ref25","doi-asserted-by":"publisher","first-page":"62","DOI":"10.1186\/s13195-015-0148-8","article-title":"Assessing clinicopathological correlation in chronic traumatic encephalopathy: rationale and methods for the UNITE study","volume":"7","author":"Mez","year":"2015","journal-title":"Alzheimers Res. Ther."},{"key":"ref26","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1007\/s00401-011-0910-3","article-title":"National Institute on Aging\u2013Alzheimer\u2019s association guidelines for the neuropathologic assessment of Alzheimer\u2019s disease: a practical approach","volume":"123","author":"Montine","year":"2012","journal-title":"Acta Neuropathol."},{"key":"ref27","doi-asserted-by":"publisher","first-page":"417","DOI":"10.1016\/j.neuroimage.2017.11.060","article-title":"Developing 3D microscopy with CLARITY on human brain tissue: towards a tool for informing and validating MRI-based histology","volume":"182","author":"Morawski","year":"2018","journal-title":"NeuroImage"},{"key":"ref28","doi-asserted-by":"publisher","first-page":"1500","DOI":"10.1016\/j.cell.2015.11.025","article-title":"Simple, scalable proteomic imaging for high-dimensional profiling of intact systems","volume":"163","author":"Murray","year":"2015","journal-title":"Cell"},{"key":"ref29","doi-asserted-by":"publisher","first-page":"3","DOI":"10.1177\/002215540505300102","article-title":"Reversing the effects of formalin fixation with Citraconic anhydride and heat: a universal antigen retrieval method","volume":"53","author":"Namimatsu","year":"2005","journal-title":"J. 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Investig."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"123","DOI":"10.1186\/s40478-023-01612-y","article-title":"Three dimensional evaluation of cerebrovascular density and branching in chronic traumatic encephalopathy","volume":"11","author":"Rosen","year":"2023","journal-title":"Acta Neuropathol. Commun."},{"key":"ref36","doi-asserted-by":"publisher","first-page":"752234","DOI":"10.3389\/fnana.2021.752234","article-title":"Comparison of different tissue clearing methods for three-dimensional reconstruction of human brain cellular anatomy using advanced imaging techniques","volume":"15","author":"Scardigli","year":"2021","journal-title":"Front. Neuroanat."},{"key":"ref37","doi-asserted-by":"publisher","first-page":"105","DOI":"10.1369\/jhc.6P7080.2006","article-title":"Standardization of immunohistochemistry for formalin-fixed, paraffin-embedded tissue sections based on the antigen-retrieval technique: from experiments to hypothesis","volume":"55","author":"Shi","year":"2007","journal-title":"J. Histochem. Cytochem."},{"key":"ref38","doi-asserted-by":"publisher","first-page":"364","DOI":"10.1038\/s41587-021-01041-z","article-title":"Highly-multiplexed volumetric mapping with Raman dye imaging and tissue clearing","volume":"40","author":"Shi","year":"2022","journal-title":"Nat. Biotechnol."},{"key":"ref39","doi-asserted-by":"publisher","first-page":"2337","DOI":"10.2147\/IJN.S297631","article-title":"Nanobody: a small antibody with big implications for tumor therapeutic strategy","volume":"16","author":"Sun","year":"2021","journal-title":"Int. J. Nanomedicine"},{"key":"ref40","doi-asserted-by":"publisher","first-page":"19","DOI":"10.1038\/s41583-019-0250-1","article-title":"Tissue clearing and its applications in neuroscience","author":"Ueda","year":"2020","journal-title":"Nat. Rev. Neurosci."},{"key":"ref41","doi-asserted-by":"publisher","first-page":"509","DOI":"10.1007\/s00401-007-0311-9","article-title":"Twenty-first century brain banking. Processing brains for research: the Columbia University methods","volume":"115","author":"Vonsattel","year":"2008","journal-title":"Acta Neuropathol."},{"key":"ref42","doi-asserted-by":"publisher","first-page":"936","DOI":"10.1016\/j.cell.2020.04.007","article-title":"The Allen mouse brain common coordinate framework: a 3D reference atlas","volume":"181","author":"Wang","year":"2020","journal-title":"Cell"},{"key":"ref43","doi-asserted-by":"publisher","first-page":"113","DOI":"10.1186\/s12915-023-01582-6","article-title":"CLARITY increases sensitivity and specificity of fluorescence immunostaining in long-term archived human brain tissue","volume":"21","author":"Woelfle","year":"2023","journal-title":"BMC Biol."},{"key":"ref44","doi-asserted-by":"publisher","first-page":"28","DOI":"10.1101\/660373","article-title":"Ultrafast immunostaining of organ-scale tissues for scalable proteomic phenotyping","author":"Yun","year":"2019","journal-title":"BioArxiv"}],"container-title":["Frontiers in Neuroscience"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fnins.2024.1474617\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T14:06:02Z","timestamp":1753884362000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fnins.2024.1474617\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,10,9]]},"references-count":44,"alternative-id":["10.3389\/fnins.2024.1474617"],"URL":"https:\/\/doi.org\/10.3389\/fnins.2024.1474617","relation":{},"ISSN":["1662-453X"],"issn-type":[{"value":"1662-453X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,10,9]]},"article-number":"1474617"}}