Fig. 6

Bee venom, Der-p1 and OVA colocalize with EEA-1 and LAMP-1 after 16 h. PDC were isolated from buffy coats by positive selection and then cultured for 16 h in the presence of IL-3 at 10 ng/ml with 40 µg/ml Alexa Fluor 555-labeled bee venom, Der p 1 or OVA. After incubation, pDC were stained with anti-CD123 APC as described. After washing with PBS, supernatants were completely aspirated, pDC resuspended in 200 µl BSA (2%) and incubated for 30 min on ice in the dark. After centrifugation at 400 g for 5 min, supernatants were completely aspirated and 100 µl fixation buffer was added. PDC were incubated for 20 min at 4 °C in the dark before washing with 1 ml permeabilization/wash buffer. Supernatants were removed and pDC resuspended in 100 µl permeabilization/wash buffer. For intracellular staining, 25 µl of either anti-EEA-1 or -LAMP-1 conjugated with Alexa Fluor 488 were added followed by incubation for 15 min at 4 °C in the dark. After washing as described, pDC were placed on dried, Poly-D-Lysine coated coverslips and incubated for 90–120 min at 37 °C. Then PBS was aspirated from the coverslips and coverslips were let dry at room temperature. DAPI for cell nucleus staining was used as described and the dried coverslips were put onto the mounting medium. Coverslips were fixed on the slide using clear nail polish and let dry. For confocal microscopy, a Leica TCS SP8 inverse confocal microscope was used, objective HC PL APO CS2 with 63 × magnification. A Colocalization of EEA-1, B of LAMP-1 with indicated allergens