Anti ifn γ antibody

The normal tissues and adjacent paracancerous and cancerous tissues were lysed with ice-cold RIPA buffer containing protease inhibitor, in accordance with the manufacturer's protocol applying the bicinchoninic acid assay (BCA) (Thermo Scientific, USA) methods for total protein, using BSA as standards. Meanwhile, the proteins were denatured at 100°C for 5 min. A total of 30 ng of proteins was separated in 12.5% SDS-PAGE and transferred to a PVDF membrane. Then, it was blocked with 5% skim milk for 2 h. The following antibodies were used in the study: rabbit anti-TIM-3 antibody (ABCAm, USA), anti-TNF-α antibody (ABCAm, USA), anti-IFN-γ antibody (ABCAm, USA), and mouse anti-β-actin antibody (Proteintech, China). The PVDF membranes were incubated with TIM-3, TNF-α, IFN-γ, and β-actin primary antibodies at 4°C overnight. The secondary antibodies (CST, USA) were used to incubate with the membranes for 1 h at room temperature. After washing with TBST, the membranes were visualized in ECL developing solution. Band intensity was scanned by ImageJ software. Quantification was determined by estimating the intensity value ratios of the reference β-actin and the target gene.

Formalin‐fixed skin tissues were cut into 4 μm‐thick sections for histological examinations. At first, the tissue sections were incubated with 3% H₂O₂ in absolute methanol to quench endogenous peroxidase. After washed three times adopting PBS, the sections were blocked using 2% goat serum. Then, the sections were incubated with specific primary antibodies overnight at 4°C, and adopted antibodies were as follows: anti‐IFN‐γ antibody (1:100, Abcam) and anti‐IL‐17 antibody (1:50, Abcam). Subsequently, the sections were washed three times employing PBS and incubated with rabbit secondary antibodies for 2 hours at room temperature. Later, the sections were stained through avidin‐biotin method using diaminobenzidine. Five high power microscopes were adopted for each tissue specimen, and the percentages of positive cells were automatically analysed by Image‐Pro Plus 6.0 software (Media cybernetics). The positivity was defined as the percentages of positively stained cells in all cells for each high power microscope, and average value for five randomly selected views was calculated for final positivity.

Purified proteins were applied on dehydrated PVDF membrane (Roche, Mannheim, Germany) in dot-blot apparatus (Bio-Rad, Hercules, CA, USA). The wells were then incubated with 200 µl blocking solution (1% BSA in TBS) for 1 h. After washing in TBST (0.05% tween 20 in TBS), the membrane was incubated for 1 h with either anti-IFNγ antibody (1∶2000; Abcam, Cambridge, UK) or anti-PPB antibody (1∶1000, custom-made, Harlan) followed by 1 h incubation with horseradish peroxidase-labeled secondary antibody. The membrane was washed again with TBST subsequent with incubation in substrate solution (0.06% diaminobenzidine (DAB), 0.08% hydrogen peroxide in TBS) for color development. The membranes were finally rinsed in distilled water and air-dried.