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.