Biotin labeled goat anti mouse igg

The ZIKV E-binding IgG responses were determined using an ELISA, as previously described (Zhao et al., 2016 (link)). Briefly, Maxisorp 96-well plates (Nunc) were coated with recombinant ZIKV E protein (Meridian Life Science Inc.) overnight at 4°C. On the next day, plates were washed extensively with PBS with 0.02% Tween 20 (PBS-T) and then blocked with 5% BSA in PBS-T for 1 h at 37°C. Serially diluted serum samples were added to the wells and incubated for 1 h at room temperature followed by washing and 1 h incubation with biotin-labeled goat anti-mouse IgG (Sigma) at room temperature. After additional washing, ZIKV E-binding IgG were detected using an HRP-conjugated streptavidin (1 h at room temperature) and tetramethylbenzidine substrate. The reaction was then stopped by addition of 1 N sulfuric acid and optical density (450 nm) measurements were determined using microplate reader (Bio-Rad). A similar ELISA was prepared with plates coated with recombinant ZIKV Eecto, DIII or DIII-LR mutant (A310E and T335K) (Zhao et al., 2016 (link)) to assess polyclonal antibody reactivity to the DIII-LR epitope. A simliar ELISA was conducted to determine serum anti-NS1 titers, with recombinant ZIKV NS1 (Native Antigen) used as the solid phase antigen.

The cells on coverslips that were treated with 100, 200 and 400 μM H₂O₂, or 85% hyperoxia for 24 h were fixed with 4% paraformaldehyde. The cells were then treated with 10% goat serum for 30 min and incubated with mouse anti-human RelA (1:2,000; cat. no. SAB1404309) and rabbit anti-human RelB (1:2,000; cat. no. SAB4300501) (both from Sigma-Aldrich, Merck KGaA) (the antibody was diluted with PBS and 5% bovine serum albumin) overnight at 4°C, then incubated with a secondary antibody [biotin-labeled goat anti-mouse IgG (cat. no. SP-9002); biotin-labeled goat anti-rabbit IgG, (cat. no. SP-9001); ZSGB-BIO, Beijing, China] for 40 min at 37°C. Finally, the cells were stained by diaminobenzidine counterstained with hematoxylin. The primary antibody was replaced with PBS as a negative control. The median absorbance values of RelA and RelB were determined using image analysis software (Prism; Shanghai, China) after scanning.