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.