Exploring Immunohistochemistry in Fish: Assessment of Antibody Reactivity by Western Immunoblotting

SIMPLE SUMMARY: In recent years, fish research has seen significant advancements, driven by the expansion of aquaculture species production, the ornamental fish industry, and biomedical studies involving aquatic organisms. Immunohistochemistry (IHC) has emerged as a valuable tool in veterinary research for studying fish biology and pathology. However, the need for validated antibodies optimized for fish species remains a challenge, leading to potential false results and misinterpretations. This study systematically assessed the reactivity of commercially available antibodies (CK AE1/AE3, vimentin, S-100, GFAP, and desmin) in IHC assays on four fish species: Sparus aurata, Dicentrarchus labrax, Oncorhynchus mykiss, and Carassius auratus. We employed Western immunoblotting (WB) and IHC techniques to evaluate antibody specificity. The results revealed a good cross-reactivity for anti-cytokeratin AE1/AE3, GFAP, and S-100 antibodies, demonstrating specific staining. Conversely, vimentin and desmin antibodies displayed no reactivity. In conclusion, this research emphasizes the need for validating antibodies specifically for fish species to ensure accurate and reliable results in fish research involving IHC analysis. ABSTRACT: In recent years, research on fish has seen remarkable advancements, especially in aquaculture, ornamental fish industry, and biomedical studies. Immunohistochemistry has become crucial in fish research, aiding in physiological and pathological investigations. However, the use of antibodies originally developed for mammals has raised concerns about their cross-reactivity and specificity in fish. This study systematically evaluated the reactivity of commonly used antibodies for diagnostic purposes, especially in fish pathology, including pan-cytokeratin, vimentin, S-100, glial fibrillary acidic protein, and desmin in the tissue of Sparus aurata, Dicentrarchus labrax, Oncorhynchus mykiss, and Carassius auratus. Western immunoblotting was employed to assess antibody specificity. The results revealed that the pan-cytokeratin and glial fibrillary acidic protein antibodies cross-react with all tested fish species, while S-100 demonstrated specific staining in sea bream, goldfish, and rainbow trout tissues. Conversely, vimentin and desmin antibodies displayed no reactivity. In conclusion, the anti-cytokeratin clone AE1/AE3 and the polyclonal rabbit anti-glial fibrillary acidic protein antibody, which are extensively used in mammals, were validated for fish immunohistochemical studies. Regrettably, D33 anti-desmin and V9 anti-vimentin clones are unsuitable for immunohistochemistry in the tested fish. These findings underscore the need for species-specific antibodies and proper validation for accurate immunohistochemistry analyses in fish research.