Establishment and evaluation of an in-situ hybridization assay for infectious pancreatic necrosis virus

In the present study, an in-situ hybridization (ISH) assay was performed on cell lines and paraffin-embedded tissue blocks infected with infectious pancreatic necrosis (IPN) virus in fish. The molecular diagnosis by ISH was established through four main steps: (1) generation of DIG (Digoxigenin)-labeled probes, (2) production of anti-DIG antibodies and their conjugation with alkaline phosphatase, (3) preparation of ISH reagents, including pre-hybridization and hybridization solutions, and (4) application of the ISH assay to cell culture and paraffin tissue sections. Protease concentrations, hybridization solutions, and assay modifications were optimized separately for cell lines and paraffin sections. Ultimately, ISH assays using DIG-labeled probes targeting the VP2 and VP3 gene regions of IPN virus were successfully performed in virus-infected cell lines and paraffin-embedded tissues.