Brief Overview of Immunohistochemistry, Its Applications and Limitations in Fish Health Investigations

Ashley L. Powell1, Alvin C. Camus1

 1Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA, 30605​

ABSTRACT

Immunohistochemistry (IHC) is a diagnostic technique routinely used in formalin-fixed paraffin-embedded tissues to identify specific cell types, pathogens, provide prognostic value, and predict response to treatment. Briefly, assays involve a target antigen within the tissue binding to a corresponding antibody and associated colorimetric or fluorometric marker. Specific steps include heat application, antigen retrieval, primary antibody preparation, secondary antibody linking, colorimetric/fluorometric labeling, and graded alcohol dehydration. IHC is relatively inexpensive, does not require specialized equipment, and is widely offered by human and animal diagnostic laboratories. Applications in fish are limited due to lack of commercially produced antibodies validated in fish and published literature describing staining characteristics in fish tissues. Interpretation of IHC slide staining results, particularly using mammalian derived antibodies, can be hindered by lack of reactivity, non-specific tissue immunoreactivity, and interspecies differences in reactivity. Given the large variation in fish species, antibodies that appropriately react in one species may not in another, therefore individual species may require unique protocol modifications. Relatively few IHC markers have been demonstrated in a select few fish species compared to the large repertoire available in domestic animal species. Pancytokeratin (AE1/AE3), a marker for epithelial cells, is perhaps the most consistent and routinely used immunohistochemical marker in teleost and elasmobranch species. S100, a nonspecific marker for Schwann cells, is used to stain neural tissues in zebrafish. Alkaline phosphatase, chromogranin A, c-KIT, desmin, estrogen receptor, neuron-specific enolase, smooth muscle actin, and vimentin, amongst others, are reported with variable success. The development of standardized and validated IHC protocols in fish is needed, particularly with the increasing use of fish as environmental sentinels, use in carcinogenesis research, and occurrence of spontaneous, poorly differentiated, or complex tumors. IHC also has application in identifying specific or families of pathogens in research or diagnostic cases. Development of immunohistochemical markers for mesenchymal tumors, such as “Hikui” in koi, is particularly needed given the morphologic similarities between neoplasms in this group: fibroma/fibrosarcoma, perivascular wall tumor, hemangiopericytoma, hemangioma/hemangiosarcoma, and peripheral nerve sheath tumor. Evaluation of various mesenchymal cell markers and protocol modifications are currently in progress.