Horseradish peroxidase hrp conjugated anti mouse antibody

Immunohistochemistry on paraffin-embedded sections was conducted, as described previously (Sahab-Negah et al. 2020 ). Briefly, brain tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The thickness of the tissues was cut into 5-μm sections. After deparaffinization, brain sections were boiled in 10 mM citric acid buffer (pH = 6) for 10 min. Sections were incubated with normal goat serum (Sigma, Germany) containing 0.3% Triton-X-100 in PBS. Sections were then incubated with primary antibodies, including mouse anti-glial fibrillary acidic protein (GFAP), a marker for reactive astrocytes (1:500; Abcam, UK); rabbit anti-ionized calcium-binding adapter molecule 1 (Iba1) antibody (1:1000; Wako) to encounter microglia at 4 °C overnight. Horseradish-peroxidase (HRP)–conjugated anti-mouse antibody (1:100; Abcam) and anti-rabbit antibody (1:500, Abcam) was added to sections at room temperature for 1 h. For negative controls, primary antibodies were deleted. The sections were visualized by 3,3′-diaminobenzidine and analyzed with a bright field microscope.

To test the immunogenicity of JEVpr/DENV2, 96-well enzyme linked immunosorbent assay (ELISA) plates were coated with 100 μl inactivated JEVpr/DENV2 in carbonate/bicarbonate buffer at pH 9.6 per well overnight at 4 °C and blocked with 1 % bovine serum albumin (BSA) at 37 °C for 2 h. The plates were washed five times with PBST (0.5 % Tween-20 in PBS), then 1:100 diluted test or negative serum in PBST were added and incubated at 37 °C for 2 h. The plates were washed five times with PBST, and then incubated with 1:5000 dilution of horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Abcam) at 37 °C for 1 h. After the final wash, HRP/substrate solution was added to each well and incubated at room temperature for 30 min. The reaction was stopped with 2 N H₂SO₄ and read at 450 nm in an ELISA Reader (Bio-Rad). A positive titer was defined as producing at least double the background absorbance.