Anti il 17 antibody

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.

Skin samples were fixed in 4% paraformaldehyde (PFA) at 4°C for 48 hours and paraffin embedded. Sections of 1.5 μm were used for hematoxylin and eosin staining to check for basic histopathologic changes. Moreover, sections were dewaxed and rehydrated and endogenous peroxidase activity was blocked by 0.1% H₂O₂ for 15 minutes. Then, sections were treated with the pepsin enzyme, incubated with Rodent Block (Biocare Medical) to reduce background, and finally incubated overnight at 4°C with the anti-CD3 antibody (1 : 100; Abcam, Cambridge, Massachusetts), anti-cytokeratin 16 antibody (1 : 100; St John's Laboratory), anti-IL-17 antibody (1 : 100; Abcam, Cambridge, Massachusetts), and anti-mouse Ly-6G/Ly-6C(Gr-1) antibody (1 : 100; R&D Systems). Then, the secondary antibody was added directly to the sections; reactions were developed in Biocare's Betazoid DAB, and nuclei were counterstained with hematoxylin. Digital images were acquired with an Olympus XC50 camera mounted on a BX51 microscope using Image-Pro Plus.