A multiplex tissue resource for high-resolution spatial protein profiling in the Human Protein Atlas.

Spatially resolved protein expression is essential for understanding tissue organization, cellular specialization, and protein function. The open-access Human Protein Atlas database (www.proteinatlas.org) has generated an extensive antibody-based tissue resource for a majority of the human protein-coding genes using conventional immunohistochemistry, enabling body-wide annotation of protein expression across normal human tissues and major cell types. However, single-marker staining often lacks the cellular and subcellular context required to resolve rare cell populations, closely related cell states, or proteins with limited functional characterization. To address this, we established a multiplex tissue resource within the Human Protein Atlas based on a large-scale multiplex immunohistochemistry workflow. The iterative workflow combines optimized antibody panels targeting established markers of cell identity, tissue organization, cellular state, and subcellular structure with candidate proteins of interest. This allows protein expression to be interpreted directly within intact tissue architecture based on expression overlap between candidate proteins and panel markers. In version 25 of the Human Protein Atlas, 1106 proteins have been analyzed using eight multiplex antibody panels across nine tissue settings, including testis, motile ciliated epithelia, salivary gland, endocrine pancreas, and kidney. These panels resolve biological contexts such as stages of spermatogenesis, Sertoli cell and ciliary subcellular compartments, salivary gland acinar and ductal structures, pancreatic endocrine cell types, and nephron segments. Here, we present the design and implementation of the multiplex tissue resource and demonstrate its utility for refining spatial protein annotation across diverse human tissue systems. By providing high-resolution spatial context for protein expression in human tissues, this publicly available resource strengthens functional protein annotation and offers a framework for generating new hypotheses about protein roles in normal tissue biology.