Zen software 2009

Confocal laser scanning microscopy images were obtained using a LSM510 Confocal microscope with an 63 × oil immersion objective (NA = 1.4, Zeiss, Germany) with appropriate laser lines (Argon laser (488 nm), Helium-Neon laser (543 nm), Argon ion laser (633), Chameleon pulsed laser (340 nm, Coherent, USA). Images were taken successively. Emission was detected using appropriate dichroic mirrors and filter sets. Images were analyzed with the ZEN software 2009 (Zeiss, Germany).

Confocal laser scanning microscopy images were obtained on a Zeiss LSM510 Confocal microscope with a 63x oil immersion objective (NA = 1.4, Zeiss, Germany). Images were taken successively, and were analyzed with the ZEN software 2009 (Zeiss, Germany).

After coculture of untouched purified monocytes with Leishmania promastigotes (MOI 10) for 20 h, 2.5 × 10⁵ cells in 30 µl were transferred to the marked reaction field of adhesion slides (Marienfeld Laboratory Glassware) prepared as recommended by the manufacturer. After cell adhesion, slides were washed twice in PBS buffer and cells were fixed with 4% Pfa. Fixed monocytes were either directly stained or additionally permeabilized with methanol (−20°C) before staining. For IL-18 staining, non-specific binding sites were blocked with PBS/2% BSA/10% normal goat serum and cells were stained with mouse-anti-IL-18 monoclonal Ab (125-2H, MBL) overnight at 4°C. As specificity control, the mouse-anti-IL-18 mAb was pretreated with rhuIL-18 (1.2 µg/ml, 30 min, 37°C). All Abs were diluted in PBS/0.5% BSA/0.5% normal goat serum. After washing with PBS/0.1% Tween Alexa Fluor 568-conjugated goat anti-mouse secondary Abs (ThermoFisher Scientific) were added for 30 min at RT. Cell nuclei were visualized by DAPI staining. Slides were mounted in Vectashield (Vector laboratories) and cover slips, dried in the dark for at least 12 h at 4°C, and analyzed by CLSFM (LSM700, Zeiss) using a 63× objective. Image processing was performed using the ZEN software 2009 (Zeiss).