Histopathology and immunohistochemistry (IHC)
Five micrometer–thick sections of the paraffin-embedded formalin-fixed tissue samples were stained with routine hematoxylin and eosin (HE) for the histopathological evaluation. All lesions observed in the HE staining tissue specimens were examined. All tissues were tested for the ASFV antigen using the IHC method. The rabbit polyclonal ASFV phosphoprotein p30 antibody (Alpha Diagnostic International, San Antonio, Texas, USA) specific to ASFV phosphoprotein p30 was used as the primary antibody. The primary antibody was diluted at 1:2500 in Blocking One (Nacalai Tesque, Japan) solution after the optimal concentration was determined. Following heat-induced antigen retrieval in citrate buffer pH6.0, the primary antibody was incubated on the tissue specimen at 4 °C overnight in a humidified chamber. Histofine® MAX PO Multi (Nichirei Biosciences, Japan) was used as the secondary antibody, and the reaction was visualized using 3,3’-diaminobenzidine (Sigma-Aldrich) chromogen. Tissue specimens collected from a pig at an ASFV-free farm were used as negative control.