Serial Immunohistochemistry for High-Dimensional Single-Cell Spatial Analysis of Human Kidney Biopsies

Yang, X.; Marlin, M. C.; Celia, A. I.; Lee, C.-Y.; Cammarata-Mouchtouris, A.; Stephens, T.; Haddad, M.; Bradshaw, L.; Saksena, D.; Buyon, J.; Izmirly, P. M.; Putterman, C.; Kamen, D.; Petri, M.; Accelerating Medicines Partnership: RA/SLE Network,; James, J. A.; Guthridge, J. M.; Fava, A.; Rosenberg, A. Z.

doi:10.64898/2026.08.06.743188

bioRxiv · 10.64898/2026.08.06.743188

Serial Immunohistochemistry for High-Dimensional Single-Cell Spatial Analysis of Human Kidney Biopsies

Yang, X.·Marlin, M. C.·Celia, A. I.·Lee, C.-Y.·Cammarata-Mouchtouris, A.·Stephens, T.·Haddad, M.·Bradshaw, L.·Saksena, D.·Buyon, J.·Izmirly, P. M.·Putterman, C.·Kamen, D.·Petri, M.·Accelerating Medicines Partnership: RA/SLE Network,·James, J. A.·Guthridge, J. M.·Fava, A.·Rosenberg, A. Z.

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Abstract

BackgroundTraditional 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. MethodsFormalin-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. ResultsThis 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. ConclusionWe 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.

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Yang, X., Marlin, M. C., Celia, A. I., Lee, C.-Y., Cammarata-Mouchtouris, A., Stephens, T., Haddad, M., Bradshaw, L., Saksena, D., Buyon, J., Izmirly, P. M., Putterman, C., Kamen, D., Petri, M., Accelerating Medicines Partnership: RA/SLE Network,, James, J. A., Guthridge, J. M., Fava, A., Rosenberg, A. Z.. 2026-08-12. Serial Immunohistochemistry for High-Dimensional Single-Cell Spatial Analysis of Human Kidney Biopsies. https://doi.org/10.64898/2026.08.06.743188

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