{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,4]],"date-time":"2026-09-04T10:49:05Z","timestamp":1788518945692,"version":"build-2803163510"},"posted":{"date-parts":[[2026,8,12]]},"group-title":"Pathology","reference-count":12,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T00:00:00Z","timestamp":1786492800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["UH2-AR067676"],"award-info":[{"award-number":["UH2-AR067676"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["UH2-AR067677"],"award-info":[{"award-number":["UH2-AR067677"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["UH2-AR067679"],"award-info":[{"award-number":["UH2-AR067679"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["UH2-AR067681"],"award-info":[{"award-number":["UH2-AR067681"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["UH2-AR067685"],"award-info":[{"award-number":["UH2-AR067685"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["UH2-AR067688"],"award-info":[{"award-number":["UH2-AR067688"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["P30AR073750"],"award-info":[{"award-number":["P30AR073750"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["R01AR069572"],"award-info":[{"award-number":["R01AR069572"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["R01DK134625"],"award-info":[{"award-number":["R01DK134625"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100002193","name":"Lupus Foundation of America","doi-asserted-by":"crossref","award":["G-2104-01274"],"award-info":[{"award-number":["G-2104-01274"]}],"id":[{"id":"10.13039\/100002193","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,8,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Background<\/jats:title>\n                  <jats:p>Traditional immunohistochemistry (IHC) with chromogen detection has limited multiplex capacity, detecting at most 4 protein markers per tissue section simultaneously, thereby restricting comprehensive spatial analysis of valuable human biopsies. We developed and validated a robust serial IHC (sIHC) staining method to detect multiple antigens on a single kidney biopsy slide, maximizing data yield for diagnosing and studying complex kidney diseases.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>Formalin-fixed, paraffin-embedded kidney biopsy sections were subjected to repeated IHC\/imaging cycles with antibody removal using an optimized sodium dodecyl sulfate-glycerol buffer stripping protocol. Images were then co-registered, and analysis was performed using a variety of methodologies, including color deconvolution, cell segmentation, and spatial clustering.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>This optimized sIHC method successfully detected up to 20 antigens on a single slide. Combining image analysis and artificial intelligence software, for example with HALO (Indica Labs), the assay assembles high-dimensional images and enables quantitative histology and single-cell spatial analysis. Using this advanced method, we were able to identify rare cell populations, such as double-negative T cells, that are challenging to detect conventionally.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion<\/jats:title>\n                  <jats:p>We have developed a validated, high-capacity sIHC protocol that uses standard IHC procedures with commercially available, clinically validated off-the-shelf antibodies. This method is a valuable, cost-effective tool for obtaining extensive, high-dimensional single-cell-resolved spatial data from limited pathology samples, such as a human kidney biopsy.<\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.08.06.743188","type":"posted-content","created":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T14:45:11Z","timestamp":1786545911000},"source":"Crossref","is-referenced-by-count":1,"title":["Serial Immunohistochemistry for High-Dimensional Single-Cell Spatial Analysis of Human Kidney Biopsies"],"prefix":"10.64898","author":[{"given":"Xiaoping","family":"Yang","sequence":"first","affiliation":[{"name":"Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0453-5355","authenticated-orcid":false,"given":"M. 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