Ab136668

IHC was conducted using Avidin-Biotinylated-Horseradish Peroxidase kits (Vector Laboratories, Burlingame, CA, USA) and DAB-Detection System (Golden Bridge International Labs, Bothell, WA, USA). After deparaffinization, the sections were rehydrated and boiled at 121°C for 10 minutes in 100 mM citrate buffer (pH 6.0) (Dako, Santa Clara, CA, USA) for heat-induced epitope retrieval. The sections were treated and incubated with primary antibodies and biotin-conjugated secondary antibodies. We used primary antibodies for human sinonasal tissues with IL-17A (1:200, rabbit immunoglobulin [Ig]G, ab136668; Abcam, Cambridge, UK), IL-23 (1:200, rabbit IgG, ab115759; Abcam), TNF-α (1:100, rabbit IgG, ab6671; Abcam), and p-mTOR (1:100, rabbit IgG, 2976; Cell Signaling Technology, Danvers, MA, USA). Mouse mucosal tissues were stained using primary antibodies such as IL-17A (1:50, rat IgG1, LS-B4912; LSBio, Seattle, WA, USA), CD68 (1:50, mouse IgG1k, MA5-13324; Invitrogen, Carlsbad, CA, USA), and Neutrophil (NIMP-R14, 1:50, rat IgG2b, ab2557; Abcam). No primary antibody control and/or isotype control were used for reagent control.

Total protein was extracted from cell lines or tissues using RIPA lysis buffer (Beyotime, Shanghai, China). Total protein was quantified using the bicinchoninic acid protein kit (Thermo Fisher Scientific). Protein (40 µg per lane) was separated with 10% SDS-PAGE and transferred onto PVDF membranes (Thermo Fisher Scientific). Subsequently, the membranes were blocked with 5% skimmed milk in TBST for 1 h at room temperature. The membranes were incubated overnight at 4°C with primary antibodies against: IL-6 (Abcam; ab239007, 1 : 1000), IL-1β (Abcam; ab118973, 1 : 1000), TNF-α (Abcam; ab219620, 1 : 1000), LC3 (Abcam; ab136668, 1 : 1000), p62 (Abcam; ab109330, 1 : 1000), Tim-1 (Abcam; ab5666, 1 : 1000) and GAPDH (Abcam; ab179467, 1 : 1000). Then, the membranes were incubated with HRP-conjugated secondary antibodies (Abcam; ab7356, 1 : 5000) for 1 h. Protein bands were visualized using the ECL kit (Thermo Fisher Scientific). β-actin was used as the loading control. IPP 6.0 (Image-Pro Plus 6.0) was used for the densitometry analysis.