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

Authors: Wiarda, Jayne; ZANELLA, ERALDO - Oak Ridge Institute For Science And Education (ORISE); Shircliff, Adrienne; Cassmann, Eric; Loving, Crystal; Devries, Alexandra; Palmer, Mitchell

Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/11/2024
Publication Date: 12/12/2024
Citation: Wiarda, J.E., Zanella, E.L., Shircliff, A.L., Cassmann, E.D., Loving, C.L., Devries, A.C., Palmer, M.V. 2024. In situ staining with antibodies cross-reactive in pigs, cattle, and white-tailed deer facilitates understanding of biological tissue status and immunopathology . Veterinary Immunology and Immunopathology. https://doi.org/10.1016/j.vetimm.2024.110865.
DOI: https://doi.org/10.1016/j.vetimm.2024.110865

Interpretive Summary: Veterinary research is stunted by the inability to identify functionally-relevant cells in tissue due to unavailability of commercially available reagents needed for detection of cell type-specific markers. To combat this issue, we tested several commercially available antibodies for cross-reactivity in three veterinary species, pigs, cattle, and white-tailed deer, and were able to successfully detect several cell type-specific markers across all three species. Detection of cell type markers was performed in tissues from all three species at an individual level, as well as through combinatorial staining of multiple markers at once. Combinatorial staining was further performed to identify which cell types become infected with Senecavirus A in pig tonsil. In total, the work provides important methodology for future veterinary research and further resolves understanding of host-pathogen interactions in pigs infected with Senecavirus A.

Technical Abstract: 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), CD3e (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/CD3e/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/CD3e/Pax5/Ki-67 allowed detection of colocalizing signals and identification of active germinal centers. Antibody compatibility with RNAscope staining was confirmed for all antibodies in all species, allowing detection of additional 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.