Splink detection kit

The experiment was performed using the streptavidin peroxidase method (SPlink detection kit; OriGene Technologies) according to the manufacturer's instructions. Paraffin-embedded intestinal tissue sections were deparaffinized, rehydrated, blocked with 10% goat serum and treated with 3% H₂O₂. Then the sections were incubated overnight at 4°C with IL-17D antibody (dilution 1:400, cat. no. bs-2612R, Bioss) or Nrf2 antibody (dilution 1:1,600, cat. no. ab 31163, Abcam), or keap1 antibody (dilution 1:1,600, cat. no. ab139729, Abcam). The sections were then incubated with biotin-labeled goat anti-rabbit IgG secondary antibody and streptavidin-horseradish peroxidase for 30 min at 37°C. Finally, the sections were developed with 3,3′-diaminobenzidine and counterstained with hematoxylin. All immunostained sections were viewed using a digital camera Nikon Eclipse E800 (Olympus Corp.). Semiquantitative analysis of the optical density (OD) values of the positive staining in the villi of each section at ×200 magnification was performed using ImageJ 6.0 (National Institutes of Health). The absorbance values of Nrf2, keap1 and IL-17D were compared via Prism Graph version 5.0 software (GraphPad Software, Inc.) after scanning.

Immunocytochemistry was performed with the SPlink Detection kit (cat no. SP-9001; OriGene Technologies, Inc., Beijing, China) and the working solutions provided by the manufacturer were used if not otherwise specified. Cell climbing slides (diameter, 8 mm) were removed from DMEM/F12 and washed three times in PBS and subsequently fixed in 4% paraformaldehyde (Beijing Solarbio Science and Technology, Ltd.) for 15 min at room temperature. Following three washes with PBS, slides were blocked with normal goat serum at 37°C for 30 min. The slides were washed in PBS and incubated with P2X₇ primary antibody (cat no. APR-004-AO; 1:200; Alomone Labs, Jerusalem, Israel) overnight at 4°C. Slides were washed in PBS and incubated with Biotin labeled goat anti-rabbit IgG polymer secondary antibody for 15 min at 37°C. Slides were washed again with PBS prior to incubation with alkaline phosphatase-labeled streptavidin for 15 min at 37°C. Slides were subsequently stained with 3,3′-diaminobenzindine solution (OriGene Technologies, Inc.) at room temperature for 10 min and sealed by neutral balsam (OriGene Technologies, Inc.). The expression of P2X₇ receptors was visualized with a fluorescence inverted microscope (magnification, ×200) and the integrated optical density (IOD) of the P2X₇ receptors was calculated using Image-Pro Plus v6.0 (Media Cybernetics Inc., Rockville, MD, USA).

The paraffin-embedded lungs (4-μm sections) were deparaffinized in dimethylbenzene and hydrated in ethanol. The tissue preparations were boiled for 7 min in a microwave oven and then cooled to room temperature. Immunohistochemistry was performed using the streptav-idin-peroxidase method (cat. no. SP-9001; SPlink detection kit; OriGene Technologies, Inc., Beijing, China) according to the kit protocol. Rabbit polyclonal anti-EPHB4 antibody was added at 4°C (1:250, overnight). Rabbit monoclonal anti-EFNB2 was added at 4°C (1:200, overnight). The sections were counterstained with hematoxylin (cat. no. G1080; Solarbio Science & Technology Co., Ltd.) for 90 sec. Negative controls were performed without primary antibody. Images of 60 slides were captured and semi-quantitative analysis was performed using Image Pro-Plus 6.0 (Media Cybernetics, Inc.).