Robust volumetric multiplex staining of centimeter-scale FFPE tissues guided by neural network-based optimization

Lin, Y.-H.; Huang, C.-Y.; Chen, Y.-H.; Xu, Z.-W.; Ko, P.-L.; Hsu, H.-H.; Tung, Y.-C.; Chen, Y.-F.; Chen, H.-C.; Chiang, A.-S.; Fiock, K. L.; Wang, K.-C.; Lin, C.-H.; Hu, S.-H.; Chen, B.-C.; Chu, L.-A.

doi:10.64898/2026.03.09.710440

bioRxiv · 10.64898/2026.03.09.710440

Robust volumetric multiplex staining of centimeter-scale FFPE tissues guided by neural network-based optimization

Lin, Y.-H.·Huang, C.-Y.·Chen, Y.-H.·Xu, Z.-W.·Ko, P.-L.·Hsu, H.-H.·Tung, Y.-C.·Chen, Y.-F.·Chen, H.-C.·Chiang, A.-S.·Fiock, K. L.·Wang, K.-C.·Lin, C.-H.·Hu, S.-H.·Chen, B.-C.·Chu, L.-A.

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Abstract

Archived formalin-fixed, paraffin-embedded (FFPE) tissues, including brain-bank specimens, are valuable resources for disease research, but rapid cyclic immunolabeling of intact specimens remains challenging. Here, we present FIDELITY, an epoxy-free workflow that balances optical transparency, immunostaining performance, and tissue integrity for multi-cycle protein profiling in centimeter-thick FFPE specimens. Neural network-guided optimization identified an SDS-glycine formulation that integrates delipidation and epitope retrieval while preserving tissue stiffness, replacing prolonged epoxy reinforcement and subsequent high-temperature antigen retrieval with a single aqueous pretreatment. Application to human Alzheimer's disease specimens enabled volumetric imaging and quantitative analysis while retaining compatibility with conventional histopathology. Its compatibility with active electrophoretic staining further supported rapid labeling and five rounds of immunolabeling in a whole FFPE mouse brain. Two-round labeling of a human glioma specimen further demonstrated the applicability of the cyclic workflow to human pathological tissue. Together, FIDELITY provides a practical platform for large-volume, multiplexed protein profiling of intact FFPE specimens.

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Lin, Y.-H., Huang, C.-Y., Chen, Y.-H., Xu, Z.-W., Ko, P.-L., Hsu, H.-H., Tung, Y.-C., Chen, Y.-F., Chen, H.-C., Chiang, A.-S., Fiock, K. L., Wang, K.-C., Lin, C.-H., Hu, S.-H., Chen, B.-C., Chu, L.-A.. 2026-03-11. Robust volumetric multiplex staining of centimeter-scale FFPE tissues guided by neural network-based optimization. https://doi.org/10.64898/2026.03.09.710440

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