Fixation perm kit

Emigrated skin DC were collected 4 days after the start of skin explant cultures. Cells were permeabilized and stained with the BD-Fixation/Perm kit (BD Biosciences, San Diego, CA, USA) according to the manufacturer's instruction. Targeting mAbs against Langerin and DEC-205 were detected with anti-mouse-Ig-APC (BioLegend), followed by blocking with 100 μg/ml mouse gamma globulin for 5 min (Jackson ImmunoResearch Laboratories) and staining of DC with anti-CD1a-FITC (clone HI149, BD Biosciences), anti-CD163-PerCP-Cy5.5 (clone GHI/61, BioLegend), anti-CD14-PE (clone HCD14, BioLegend), anti-HLA-DR-PE-Cy7 (clone L243, BioLegend) for 10 min at 4°C. Appropriate isotype controls from Biolegend were used for FACS stainings to confirm specific staining. Analyses were performed on a FACS Calibur and Canto II instrument (BD Biosciences). The percentage of targeted skin DC was determined by pregating on viable HLA-DR⁺ cells, followed by gating for the various skin DC subsets with CD1a and CD14. In the three different skin DC subsets, cells positive for the targeting mAb were determined by setting the gate on DC from skin explants injected with isotype controls.