Ab53707

Lesional skin biopsy specimens from AD patients or OXA-induced mouse tissue specimens were fixed with a 12% formaldehyde solution and embedded in paraffin. For immunofluorescence staining, cells or skin biopsy specimens were incubated with the corresponding primary antibodies at 4°C overnight as follows: anti-K17 (1:1000; ab53707; Abcam), anti-CCL20 (1:300; ab9829; Abcam), anti-CD3 (1:500; ab16669; Abcam), anti-CD4 (1:500; ab183685; Abcam), anti-CD8 (1:500; ab22378; Abcam), anti-STAT3 (1:500; #124H6; Cell Signaling Technology) and anti-p-STAT3 (1:100; #9145; Cell Signaling Technology). After three washes with PBS, Cy3- or fluorescein isothiocyanate-conjugated secondary antibodies (1:200; BioLegend) were added, and Hoechst 33258 (Solarbio Technology, Beijing, China) was applied to label the nuclei. Samples were detected by a confocal microscope (LSM880; Carl Zeiss). For IHC staining, samples were incubated with 0.3% H₂O₂ for 10 min prior to staining with the corresponding primary antibodies: anti-K17 (1:1000; ab53707; Abcam) and anti-CCL20 (1:300; ab9829; Abcam) at 4°C overnight. Sections were subsequently incubated with an HRP-labeled goat anti-mouse/rabbit antibody (CoWin Biosciences) for 1 h at room temperature. 3,3’-Diaminobenzidine (Gene Tech, Shanghai, China) was used to detect biotinylated antibodies.

Biopsies obtained from the lesional skin of psoriasis patients and the skin of healthy controls or from IMQ-induced mice were fixed with a 12% formaldehyde solution and embedded in paraffin. For immunofluorescence staining, cells or skin biopsy specimens were incubated with the following primary antibodies overnight at 4°C: anti-K17 (1:1000, ab53707, Abcam, UK), anti-ENO1 (1:400, ab227978, Abcam, UK), and anti-Ki67 (1:1000, ab15580, Abcam, UK). After three washes with PBS, the cells were incubated with Cy3- or FITC-conjugated secondary antibodies (1:200, BioLegend, San Diego, USA), and Hoechst 33258 (1:1000, Solarbio Technology, China) was applied to all cells to label the nuclei. Samples were analyzed using a confocal microscope (LSM880, Carl Zeiss, Germany). For immunohistochemical staining, the tissue sections were incubated with 0.3% H₂O₂ for 10 min and then incubated with one of the following primary antibodies: anti-K17 (1:1000, ab53707, Abcam, UK) or anti-ENO1 (1:400, ab227978, Abcam, UK) overnight at 4°C. Sections were subsequently incubated with an HRP-labeled goat anti-mouse/rabbit antibody (CWBIO, Peking, China) for 1 h at room temperature. DAB (Gene Tech, Shanghai, China) was used to detect biotinylated antibodies.

Cultured cells were washed once with PBS and fixed in 4% paraformaldehyde (Electron Microscopy Sciences) for 10 min at RT. Samples were washed twice with PBS, and 0.1% Triton-X solution was added to promote membrane permeability by the antibody against cytokeratin 17. The samples were washed three times and then treated with CAS-Block solution (008120, Thermo Fisher Scientific, MA, U.S.A.) for 30 min at RT. Primary antibodies were diluted with CAS-Block solution and then applied to the samples, and the treated samples were incubated at 4 °C overnight. We used the following antibodies at a 1:100 dilution: anti-cytokeratin 17 antibody (ab53707, Abcam) and anti-CD99 antibody [EPR3096] (ab108297, Abcam), a 1:50 dilution: EZH2 antibody (21,800–1-AP, proteintech). After rinsing four times, for 15 min each time, in PBS, the samples were incubated at room temperature for 3 h with fluorescent secondary antibodies (Alexa-488, Invitrogen, CA, U.S.A. or CoraLite488, proteintech, IL, U.S.A.). The samples were observed under a fluorescence microscope after four final rinses, each for 15 min each time, in PBS.