In situ staining with antibodies cross-reactive in pigs, cattle, and white-tailed deer facilitates understanding of biological tissue status and immunopathology.

Identifying cellular markers within archived formalin-fixed, paraffin-embedded (FFPE) tissues is critical for understanding tissue landscapes impacting animal health, but in situ detection methods are limited in veterinary species by a restricted toolbox of species-compatible immunoreagents. We identify antibodies with conserved in situ reactivity to IBA-1 (macrophages/dendritic cells), CD3ε (T cells), Pax5 (B cells), Ki-67 (cycling cells), and cytokeratin type I/II (epithelial cells) in FFPE tissues of pigs, cattle, and white-tailed deer. Multiplexed brightfield detection (IBA-1/CD3ε/Pax5) in lymph nodes of all three species demonstrated species-specific and species-conserved features of cellular architecture. Multiplexed fluorescent staining in pig lymph nodes for IBA-1/CD3ε/Pax5/Ki-67 allowed detection of colocalizing signals and identification of active germinal centers. Antibody compatibility with RNA in situ hybridization was confirmed for all antibodies in all species, allowing co-detection of RNA markers, which is a strategy highly useful in veterinary species where protein-reactive reagents are often lacking. Multiplexed protein and RNA staining was performed in tonsil tissue of a pig infected with Senecavirus A, enabling identification of virally-infected cell types via simultaneous detection of host cell type-specific proteins and virus-specific RNA. Findings have important applications for future in situ identification and comparative study of tissue landscapes and immunopathology in a diverse range of veterinary species.