Immunohistochemical Analysis of Rabies Virus Protein

Brain sections were prepared as described previously (36 (link)). After antigen retrieval in citrate buffer for 5 min using a pressure cooker, the sections were treated with 0.3% H₂O₂ in methanol for 15 min to inactivate endogenous peroxidase. The sections were then treated with 10% goat serum (Nichirei Biosciences) for 1 h at room temperature and incubated at 4°C overnight with the primary antibody against RABV N (code PA321352LA01RAI; Cusabio; 1:2,500 dilution). After three washes with 0.01% PBST, secondary staining was performed with EnVision system, HRP-labeled polymer anti-rabbit (catalog no. K4220; Dako) for 1 h at room temperature. The slides were washed three times with PBST, and a 3,3′-diaminobenzidine (DAB) substrate kit (catalog no. 425011; Nichirei) was used to visualize the immunostaining.