D eclipse confocal laser scanning microscope

NHEKs (2 × 10⁴) cultured on slides (Lab-Tek, Rochester, NY, USA) with or without IL-4, FICZ, and/or Glyteer were washed in phosphate-buffered saline (PBS), fixed with acetone for 10 min, and blocked using 10% bovine serum albumin in PBS for 30 min. The samples were incubated with an anti-FLG antibody or anti-OVOL1 antibody in WesternBreeze Blocker/Diluent (Invitrogen, Carlsbad, CA, USA) overnight at 4 °C. The slides were washed with PBS before incubation for 1 h at room temperature with a secondary antibody: an anti-rabbit IgG or anti-mouse IgG antibody (conjugated with Alexa Fluor 488 or Alexa Fluor 546; Molecular Probes, Eugene, OR, USA). After nuclear staining with DAPI, which emits blue fluorescence, slides were mounted with the UltraCruz Mounting Medium (Santa Cruz Biotechnology). The nuclear translocation of OVOL1 in NHEKs was evaluated by detection of the coincidence of the two-color fluorescence (green and blue) in the nucleus. All the samples were analyzed using a D-Eclipse confocal laser scanning microscope (Nikon, Tokyo, Japan).

NHEKs were cultured on slides (Lab-Tek, Rochester, NY, USA). These slides were then washed in phosphate-buffered saline (PBS), fixed with acetone for 10 min, and blocked using 10% bovine serum albumin (Roche Diagnostics, Basel, Switzerland) in PBS for 30 min for the staining of IL-37. Samples were incubated with primary anti-human IL-37 polyclonal rabbit antibody (1:100) (Abcam) or IgG rabbit polyclonal antibody (Abcam) in Western Breeze Blocker/Diluent (Thermo Fisher Scientific) overnight at 4°C. The slides were then washed with PBS before incubation with anti-rabbit secondary antibody (Alexa Fluor 546; Molecular Probes, Eugene, OR, USA) for 1 h at room temperature. After nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI), the slides were mounted with UltraCruz mounting medium (Santa Cruz Biotechnology, Dallas, TX, USA). All samples were analyzed using a D-Eclipse confocal laser scanning microscope (Nikon, Tokyo, Japan).

Image-iT LIVE Green Reactive Oxygen Species Detection Kit (Thermo Fisher Scientific) was used following the manufacturer’s instructions. In brief, this involved the use of DCFH-DA, a cell-permeable non-fluorescent probe that is de-esterified intracellularly and oxidized to highly fluorescent 2′,7′-dichlorofluorescein in the presence of ROS. NHEKs were treated with tapinarof or benzo(a)pyrene for 6 h, and then washed and incubated with DCFH-DA (25 µM) for 30 min at 37 °C. The fluorescent signal of 2′,7′-dichlorofluorescein, the oxidation product of DCFH-DA, was analyzed using a D-Eclipse confocal laser scanning microscope (Nikon, Tokyo, Japan).

Immunofluorescent analysis was performed on cell sheets cultured in 4-well slide chambers (Lab-Tek, Rochester, NY, USA) with KGM-Gold (Lonza) for 48 h, scratched using 1000-μL tips (Greiner Bio-One), and incubated for 6 h at 37 °C in 5% CO₂. The cells were washed with phosphate-buffered saline 3 times for 5 min each and fixed in cold acetone for 10 min at room temperature. The cell sheets were blocked with 10% bovine serum albumin (Roche Diagnostics, Basel, Switzerland) and incubated with mouse monoclonal anti-IL13Rα2 (Abcam) or control normal mouse IgG (Santa Cruz Biotechnology). Goat anti-mouse IgG conjugated with Alexa Fluor 488 dye (Thermo Fisher Scientific) was used for the secondary antibody. The nucleus was stained with 4′,6-diamino-2-phenylindole (DAPI). Slides were then mounted with Ultra Cruz mounting medium (Santa Cruz Biotechnology) and were observed using a D-Eclipse confocal laser scanning microscope (Nikon, Tokyo, Japan). The immunofluorescence intensity was measured using ImageJ software.

Immunofluorescence analysis was performed as reported previously [61 (link)]. The cells were cultured in 4-well slide chambers (1.5 × 10⁵ cells/well) (Lab-Tek, Rochester, NY, USA) in accordance with the culture methods described herein. Then, the cell sheets were scratched and incubated for 6 h at 37 °C in 5% CO₂. The cell sheets were washed with PBS 3 times for 5 min each and fixed in cold acetone for 10 min at room temperature. The cell sheets were blocked with 10% BSA (Roche Diagnostics, Basel, Switzerland) and incubated with rabbit anti-human CCL20 polyclonal antibody or control normal rabbit polyclonal IgG. Goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody with Alexa Fluor 488 dye was used as the secondary antibody. The nucleus was stained with 4′,6-diamino-2-phenylindole (DAPI). Slides were then mounted with Ultra Cruz mounting medium (Santa Cruz Biotechnology, Dallas, TX, USA) and were observed using a D-Eclipse confocal laser scanning microscope (Nikon, Tokyo, Japan).

Twelve-well plates were seeded with HaCaT cells. Experimental procedures were carried out as described above. After a PBS rinsing and a 20 min fixation with 4% paraformaldehyde at room temperature, the cells were fixed for another 20 min at room temperature. Cells were permeabilized in 0.1% Triton X-100 through PBS for 15 min after fixation. After an hour, the cells were blocked using PBS containing 5% BSA. The cells were treated with a diluted primary antibody overnight at 4 °C. The material was colored with DAPI for 10 min at 37 °C after two hours of incubation with the secondary antibody. Analysis of all samples was performed using a Nikon D-Eclipse confocal laser scanning microscope (Tokyo, Japan). AffiniPure Donkey anti-rabbit or anti-mouse IgG (H + L) and Alexa Fluor^® 488/Cy3 antibodies (Jackson Immuno Research USA, West Grove, PA, USA) were used to detect anti-CCL17 and anti-CCL22 antibodies.

NHEKs (2 × 10⁴) were cultured on slides (Lab-Tek, Rochester, NY, USA) with or without RCE for 5 h. The slides were then washed in phosphate-buffered saline (PBS), fixed with acetone for 10 min, and blocked using 10% bovine serum albumin (Roche Diagnostics, Basel, Switzerland) in PBS for 30 min. Samples were incubated with primary anti-AHR (1:50) or anti-OVOL1 (1:50) antibody in Western breeze blocker/diluent (Invitrogen, Carlsbad, CA, USA) overnight at 4 °C. The slides were then washed with PBS before incubation with anti-rabbit secondary antibody (Alexa Fluor 546 or 488; Molecular Probes, Eugene, OR, USA) for 1 h at room temperature. After nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI), the slides were mounted with UltraCruz mounting medium (Santa Cruz Biotechnology). All samples were analyzed using a D-Eclipse confocal laser scanning microscope (Nikon, Tokyo, Japan).