Immunofluorescence and IRF3 as well as p65 nuclear translocation analysis

Primary KCs were grown on coverslips to 60% confluency, stimulated with low MW p(I:C) (20 µg/ml), fixed, and stained. Migratory LCs collected from epidermal sheet explants that were cultured without (unstimulated, control medium) or with unlabeled p(I:C) (20 µg/ml) for 24 h at 37°C were placed on adhesion slides (300 LCs in a volume of 10 µl/slot). Fixed cells were stained with antibodies directed against CD207, MAVS, IRF3, and p65. To label mitochondria, KCs and LCs were incubated with a MitoTracker (Mitochondrion-Selective Probes, dilution 1:1000; Thermo Fisher Scientific) according to the manufacturer’s instructions. The probe passively diffuses across the plasma membrane and accumulates in active mitochondria. Cells were viewed under a conventional and/or confocal microscope (data not shown). Liver hepatocellular HepG2 cells were used as a positive control for PRR staining protocols according to the antibody data sheets (data not shown).