Il 22

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).

For stimulation, HaCaT and pKC cell cultures were carried out until 70% confluence was achieved. After this time, keratinocytes were enzymatically digested using trypsin/EDTA (0.25 or 0.05% (w/v)) for HaCaT and pKC, respectively, and seeded on 6-well plates at a density of 5 × 10³ cells/cm² in serum-free medium (SFM, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) with the addition of 30 μg/mL bovine pituitary extract (BPE), 0.2 ng/mL epidermal growth factor (EGF; Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and 1% (v/v) A/A solution to a final concentration. Following a 24 h incubation period, the medium was changed to SFM without growth supplements for a further 16 h of incubation. Subsequently, cells were activated with a cytokine mix (5MIX, i.e., interleukin (IL) 1 alpha (IL-1α), IL-17A, IL-22, oncostatin M (OSM), and tumour necrosis factor alpha (TNF-α), 2 ng/mL each; Abcam, Cambridge, United Kingdom) for a further 24 h or left non-activated as a control. As an additional panel to induce keratinocyte differentiation, activated or non-activated cells with the addition of 2 mM calcium ions were also tested.