Ab203883

Proteins from brain tissues (homogenized) were extracted using RIPA buffer (Biosharp, Hefei, China) and quantified using a bicinchoninic acid kit (Biosharp, Hefei, China). The extractive was heated in boiling water for 10 min, then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% skim milk and then incubated with primary antibodies anti-Nrf2 (1: 1000; ab62352; Abcam), anti-NF-κB phosphorylated p65 (p-p65) (S536) (1: 1000; ab86299; Abcam), anti-CD16 (1: 1000; ab203883; Abcam), anti-CD11b (1: 1000; ab13357; Abcam) and anti-β-actin (1: 1000; ab8226; Abcam), respectively, followed by being gently shaken at 4 °C overnight. The membranes were then rinsed with TBST and incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Proteintech, Wuhan, China) for 1 h at room temperature. Finally, electrochemiluminescence kit (Biosharp, Hefei, China) was used for developing the protein bands. The gray value of each band was analyzed with the software ImageJ (NIH, Bethesda, MD, USA).

The expression of IL-2, PD-1, CD3, CD8, CD16 and HLA-ABC in individual PTC and their adjacent non-tumor tissues was examined by IHC. Briefly, the paraffin-embedded tissue sections (4 µm) were deparaffinized, rehydrated and treated with 3% H₂O₂in methanol, followed by heat-induced antigen retrieval (0.01mol/L citrate buffer, pH 6.0). After being blocked with 5% bovine serum albumin (BSA), the sections were incubated overnight at 4℃ with antibodies against IL-2 (26156-1-AP, Proteintech), PD-L1(ab140950, Abcam), CD3 (ab16669, Abcam), CD8 (ab17147, Abcam), CD16 (ab203883, Abcam) or HLA Class I ABC (ab70328, Abcam). After being washed, the bound antibodies were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized with 3,3'-diaminobenzidine (DAB) using a IHC kit (KIHC-1, Proteintech), followed by counterstained with hematoxylin. Images were obtained under an Olympus IX71 microscope (Olympus, Japan) and analyzed by two experienced pathologists in a blinded manner. The immunostaining was scored, according to the percentages of positive cells (0, no staining; 1, ≤10%; 2, 10‑50%; and 3, >50%) and the intensity (0, negative; 1, weak; 2, moderate; and 3, strong), and then were multiplied to get an immunostaining score (IS).