Ccl20

The concentrations of CCL2, CCL20 and CXCL 13 (Abcam, USA) in the rumor cell culture supernatants, as well as Ig G, IFN-γ and Granzyme B in the B culture supernatants were detected using commercial ELISA (BD PharNingen, USA) kits according to the manufacturer's instructions.

Gradient concentrations of xylene and alcohol were used for paraffin section dewaxing and hydration, and then the sections were heated for antigen retrieval. After cooling to room temperature, the sections were soaked in 3% H₂O₂ solution to inactivate the endogenous peroxidase (only for immunohistochemistry). All sections were treated with 10% goat serum for 30 min to block the antigen and then incubated with primary antibodies at 4°C overnight. The secondary antibody (Zhongshan Biology Co. Ltd, China) corresponding to the primary antibody was incubated at room temperature for 30 min. For immunohistochemistry staining, diaminobenzidine was used for antibody staining and hematoxylin (Beyotime, China) for nuclear staining. For immunofluorescence staining, DAPI was used for nuclear staining. The results were observed and photographed under a microscope (Olympus, Japan).
The primary antibodies used are described as follows: IL-1β (1:200, Abcam, United Kingdom); IL-6 (1:200, Abcam, United Kingdom); IL-8 (1:200, Bioworld, United States); K1 (1:200, Abcam, United Kingdom); K6 (1:200, Thermo Fisher Scientific, United States); TNF-α (1:200, Abcam, United Kingdom); IL-23 (1:200, Abcam, United Kingdom); CCL20 (1:100, Abcam, United Kingdom); IL-22; IL-17 (1:250, Abcam, United Kingdom); IFN-γ (1:200, Abcam, United Kingdom); and BrdU (Abcam, United Kingdom).

Skin samples were placed in 0.25% dispase II (Roche, Switzerland) solution at 4 °C for 16 h. The epidermis and dermis were separated by forceps. The epidermis was cut and put into 0.25% trypsin and 0.02% EDTA digestion (Beyotime), and the cells were cultured in the defined keratinocyte-SFM (Gibco, New York, USA) for 5 days to get primary keratinocytes. The dermis was cut into small pieces and cultured in Dulbecco’s modified eagle medium (Gibco) containing 10% fetal bovine serum (Sciencell), 1% penicillin/streptomycin (Beyotime) for 7 days to get primary fibroblast. Cells were cultured with recombinant human TNF-α protein (Abcam) for 12 h, cells cultured with medium provided a control group. Culture supernatants were collected to measure the levels of cytokines secreted by keratinocytes and fibroblast. The levels of CXCL8 (Raybiotech, Georgia, USA), CCL20 (Abcam), IL-6 (Invitrogen), IL-1B (Abcam), and IL-17B (Novus Biologicals, Colorado, USA) were determined by ELISA according to the manufacturer’s instructions.