4.3. Histology and Immunohistochemistry

Histopathology was performed by an experienced veterinary pathologist, blinded to both gross lesions as well as to the patient’s signalment and history. Formalin-fixed samples were sent to the Department of Comparative Biomedicine and Food Science of the University of Padua. Sections (4 μm) obtained from formalin-fixed paraffin-embedded samples were stained with hematoxylin–eosin for histology with an automated stainer (Autostainer XL, Leica Biosystems, Wetzlar, Germany). For immunohistochemistry (IHC), 4 μm paraffin sections were placed on surface-coated slides (Superfrost Plus, ThermoFisher Scientific, Milan, Italy). Immunostaining was performed with an automatic immunostainer (Ventana Benchmark XT, Ventana Medical System, Tucson, AZ, USA). Antigen retrieval was performed with standard CC1 (Heat Induced Epitope Retrieval, HIER, in Tris-EDTA buffer pH 7 at 95 °C for 44 min). As the primary antibody, a mouse monoclonal antibody against the feline coronavirus was used (dilution 1:500, clone FIPV3-70 Serotec, Oxford, UK). After incubation at 37 °C for 30 min, a kit with a secondary antibody with horseradish peroxidase (HRP)-conjugated polymer that binds to mouse and rabbit primary antibodies (ultraViews Universal DAB, Ventana Medical System, Tucson, AZ, USA) was used. All reagents were dispensed automatically except for the primary antibody, which was manually dispensed. FIP positive tissues were used as internal positive controls. For negative controls, the antibody diluent (Ventana Medical Systems) was applied instead of the primary monoclonal antibodies.