Anti nak tbk1

Formalin-fixed, paraffin-embedded tissue sections were sectioned at a thickness of 4 μm, and then routinely deparaffinized in xylene and rehydrated in graded alcohol solutions. Antigen retrieval was performed with sodium citrate buffer (10 mM, PH = 6.0) using microwaves for 20 min. Prior to incubation with the primary antibodies, all the sections were treated with 3% H₂O₂ for 20 min at room temperature to block endogenous peroxidase activities. Sections were co-incubated with 5% BSA buffer for 1 h to attenuate non-specific protein binding. Then, slides were incubated with anti-γδ TCR (1:10, Thermo Fisher, Waltham, MA, Catalog #5A6.E9), anti-STING (1:200, Cell Signaling Technology, Danvers, MA, USA, Catalog #D2P2F) and anti-NAK/TBK1 (1:250, Abcam, Cambridge, UK, Catalog #EP611Y) primary antibodies in a moist chamber at 4 °C overnight, separately. Blank controls were treated with phosphate buffered saline (PBS) buffer instead. Then, HRP polymer-conjugated secondary antibodies were used for 1 h at 37 °C. The slides were visualized with diaminobenzidine (DAB) solution, followed by hematoxylin counterstaining. The positive staining was measured from at least 4 randomly selected areas at 400× magnification using integrated optical density (IOD) by Image Pro Plus 6.0 (Media Cybernetics, Inc., Silver Spring, MD, USA).

Fresh tissue samples from patients with OLP and healthy controls were carefully dissected. Protein concentrations were measured according to a BCA assay (Thermo Scientifc, Waltham, MA, USA) generated standard curve. A total of 40 µg of protein was denatured and then subjected to 12% SDS-polyacrylamide gel electrophoresis, followed by transfer onto polyvinylidene fuoride membranes (Millipore Corporation, Billerica, MA, USA). The membranes were incubated with primary antibodies (anti-γδ TCR (Thermo Fisher, Catalog #5A6.E9), anti-STING (Cell Signaling Technology, Catalog #D2P2F), anti-NAK/TBK1 (Abcam, Catalog #EP611Y), anti-p-STING (Cell Signaling Technology, Catalog #E9A9K), anti-p-TBK1 (Cell Signaling Technology, Catalog #D52C2), anti-IFN-γ (Abclonal, Wuhan, China, Catalog #A12450), anti-IL-17A (Bioworld, Dublin, OH, USA, Catalog #BS6041), anti-HLA-DR (Nonus, CO, USA, Catalog #NBP2-67610) and anti-CCR6 (Bioss, Beijing, China, Catalog #BS-1542R)). After being washed, the membranes were then probed with secondary antibodies. Next, the blots were stained using an enhanced chemiluminescence detection kit (Applygen, Beijing, China). The relative protein levels were calculated based on β-actin (Proteintech, Chicago, IL, USA, Catalog #66009-1-Ig) as the loading control and were densitometrically analyzed by Image J software (NIH, Bethesda, MD, USA).