Fitc conjugated il 17a

The samples of imiquimod-applied mouse back skin were harvested. They were formalin-fixed and stained with hematoxylin and eosin. For immunohistochemistry, the mouse skin was embedded in an optimal cutting compound, snap-frozen in liquid nitrogen and stored at −80 °C. Cryosections were fixed with cold acetone for 5 min and incubated overnight at 4 °C with anti-mouse MHC class II antibody (1/50 dilution, Abcam, Cambridge, UK) and anti-mouse CD3 antibody (1/100 dilution, Abcam). Tissues were subsequently stained with an avidin-biotin peroxidase complex using a Vector ABC staining kit (Vector Laboratories, Burlingame, CA). Diaminobenzidine was used for visualizing the staining, and counterstaining with Mayer hematoxylin was performed according to the manufacturers’ instructions. Stained cells were counted in 10 random grids under high original magnification (×400) power fields of a light microscope. Each section was examined independently by two investigators in a blinded manner. In some experiments, 5 μm-thick tissue sections from freshly frozen samples were stained with fluorescein isothiocyanate (FITC)-conjugated IL-17A (BioLegend, San Diego, CA, USA) and FITC-conjugated IFN-α (Biorbyt, Cambridge, UK), or FITC-conjugated IL-10 (BioLegend).

The frequency of Th17 and Tregs in CD4+ T cells in peripheral blood were determined with the use of flow cytometry as described previously with some modifications (Chi et al., 2007 (link), 2010 (link)). Briefly, 200 μl of fresh peripheral blood obtained from caudal vein of each rat. The red blood cells were lysed and washed with PBS. Cells were initially stained extracellularly with use of phycoerythrin (PE) anti-human CD4 (eBioscience, United States) at 4°C for 20 min. Subsequently, cells were fixed and permeabilized, and stained with fluorescein isothiocyanate (FITC)-conjugated IL-17A (BioLegend, United States) for Th17 detection and PerCPCy5.5-conjugated IFN-γ (BioLegend, United States) for Th1 detection. For Tregs, the CD4-PE cells were stained with FITC-conjugated CD25 (BioLegend, United States). Flow cytometric analysis was performed with use of a fluorescence-activated cell sorter Calibur cytometer. Data were analyzed with CellQuest software (Becton Dickinson, United States).