Ta324190

FFPE human skin tissues were sectioned and stained as follows: sections were deparaffinized; rehydrated; and heated at 100°C for 20 minutes in antigen retrieval buffer, pH 6, for treatment with IFN-α, IFN-β, and IL-18, whereas antigen retrieval buffer, pH 9, for IFN-κ and MX-1 was used. Slides were washed, treated with 3% hydrogen peroxide in PBS for 5 minutes, blocked, and incubated with anti–IFN-α, anti–IFN-β, anti–IFN-κ, anti-MX1, and anti-IL18 antibodies at 1:100 dilutions (Santa Cruz Biotechnology sc-80996, Abcam ab140211, Abnova H00056832, Abcam ab95926, and ORIGENE TA324190, respectively) overnight at 4°C. Appropriate negative (no primary or secondary antibodies or isotype control antibodies IgG [Abcam 125938], IgG2ak [Invitrogen, Thermo Fisher Scientific 14-4724-82], and IgG2bk [BioLegend 401201]) antibodies were stained in parallel with each set of the previously mentioned slides. All slides were then incubated with biotinylated secondary antibodies at 1:200 dilutions (Vector Laboratories goat anti–rabbit IgG biotinylated antibody PK-6101 and anti–mouse IgG biotinylated antibody PK-6102), followed by incubation with Vectastain ABC reagent, and stained with peroxidase substrate, counterstained with hematoxylin, dehydrated, and mounted. Images were acquired using a Zeiss microscope at indicated magnifications.

Protein expression was analyzed by western blotting as previously described (Chai et al. 2014 (link); Yuan et al. 2015 (link)). For western blot analysis, primary antibodies against NFκB (rabbit polyclonal antibody, ab16502), TNFα (rabbit polyclonal antibody, ab6671), NLRP3 (rabbit polyclonal antibody, ab210491), IL-1β (rabbit polyclonal antibody, ab9722), IL-6 (rabbit polyclonal antibody, ab208113) and caspase1 (rabbit polyclonal antibody, ab1872) were purchased from Abcam (UK). TLR4 (mouse monoclonal antibody, sc-293072) and ASC (mouse monoclonal antibody, sc-271054) were purchased from Santa Cruz (USA). IL-18 (rabbit polyclonal antibody, TA324190) was purchased from ORIGENE (USA). The goat anti-mouse IgG-HRP (ZB2305) and goat anti-rabbit (ZB2301) IgG-HRP secondary antibodies were purchased from ZSGB-Bio, Beijing, China. The targeted proteins were visualized with the Super Signal West Femto Chemiluminescent Substrate (Thermo Scientific Pierce) and the intensity of the visualized bands were analyzed using Quantity One software (Bio-Rad). β-actin (mouse monoclonal antibody, TA-09, Zhongshan Jinqiao Biotech company, Beijing, China) was used as an internal control. Data are expressed as a ratio to β-actin.