2.8 Immunofluorescence staining
We performed immunofluorescence staining for IL-35 and M-MDSCs in the skin of patients with psoriasis and healthy controls and MDSCs and iNOS+MDSCs in murine skin lesions. Mice were treated with IL-35; the back-skin tissue of mice was obtained the day after the last treatment, frozen, and then stained. For staining, the primary antibodies (rat anti-human p35 and rabbit anti-mouse EBI3+ for human IL-35, rat anti-human CD14 and rabbit anti-human HLA-DR for human M-MDSCs, rat anti-mouse CD11b and rabbit anti-mouse Gr-1 for mouse MDSCs, rabbit anti-mouse Gr-1 and rat anti-mouse iNOS for mouse iNOS+MDSCs) were incubated with the samples at 4 ℃ overnight and secondary antibodies including goat anti-rabbit IgG-FITC, goat anti-rat IgG-TR or anti-rabbit IgG-TR, goat anti-rat IgG-FITC respectively for 1 h (all first antibodies were from Abcam, and second antibodies were from zsbio, Beijing, China). Tissue sections were examined under a fluorescence microscope (Olympus Optical, Tokyo, Japan), and images were captured at ×400 magnification.