Ab9829

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.

Assays were performed as previously reported [20 (link)]. In brief, for the IF experiment, cells were incubated with primary antibodies against CCL20 (1:200, #ab9829) (Abcam) at 4 °C overnight, then incubated with the fluorescent secondary antibody Alexa Fluor 594-conjugated goat anti-mouse IgG (Cell Signaling Technology) and imaged using a fluorescence microscope (DM5000B, Leica). For IHC, the paraffin sections were incubated with antibodies against CCL20 (1:200, #ab9829) (Abcam). Intensity scores were recorded as: 0 (no staining), 1 (weakly staining, light yellow), 2 (moderately staining, yellowish brown), and 3 (strongly staining, brown). In addition, we also detected the expression of ki67 (1:1000, #9449), CD68 (1:200, #97778), CD163 (1:400, #93498), CD206 (1:200, #24595) and CD31 (1:100, #77699) (Cell Signaling Technology) in PCa tissue or xenograft tissue. Images were observed under an Olympus multifunction microscope (Olympus BX51, Tokyo, Japan). All evaluations were performed by three independent senior pathologists using the same microscope.

Assays were performed as previously reported [20 (link)]. In brief, for the IF experiment, cells were incubated with primary antibodies against CCL20 (1:200, #ab9829) (Abcam) at 4 °C overnight, then incubated with the fluorescent secondary antibody Alexa Fluor 594-conjugated goat anti-mouse IgG (Cell Signaling Technology) and imaged using a fluorescence microscope (DM5000B, Leica). For IHC, the paraffin sections were incubated with antibodies against CCL20 (1:200, #ab9829) (Abcam). Intensity scores were recorded as: 0 (no staining), 1 (weakly staining, light yellow), 2 (moderately staining, yellowish brown), and 3 (strongly staining, brown). In addition, we also detected the expression of ki67 (1:1000, #9449), CD68 (1:200, #97778), CD163 (1:400, #93498), CD206 (1:200, #24595) and CD31 (1:100, #77699) (Cell Signaling Technology) in PCa tissue or xenograft tissue. Images were observed under an Olympus multifunction microscope (Olympus BX51, Tokyo, Japan). All evaluations were performed by three independent senior pathologists using the same microscope.