Ix71 inverted fluorescence phase contrast microscope

Macrophages were in vitro differentiated for 7 days on poly-l lysine coated coverslips before addition of C26:0 (100 µM) or the solvent ethanol. After 24 h of treatment, cells were washed with PBS, fixed in 3% paraformaldehyde for 20 min and again washed with PBS. For immunofluorescence, cells were permeabilized with 0.1% Triton for 5 min and blocked with 2% FCS, 2% BSA and 0.2% fish gelatin for 30 min, followed by PBS washing. For vinculin staining, cells were incubated for 2 h with a monoclonal mouse anti-human vinculin antibody (1:250, #V9264, Sigma). Filamentous (F)-actin was stained for 1 h using AlexaFluor⁴⁸⁸ Phalloidin (1:2000, ThermoFisher). Then, a secondary donkey anti-mouse IgG Cy3 antibody (1:400, #IR715-165-150, Jackson) was added for 1 h. After staining, cells were washed with PBS, nuclei stained with DAPI (1:2000) for 30 min in the dark before mounting in Mowiol. Fluorescence microscopy was carried out using an Olympus Ix71 inverted fluorescence phase contrast microscope with quantification done for 10 areas per coverslip containing approximately 130 cells. Macrophages displaying more than five podosomes were counted as podosome positive. For visualization of podosomes by confocal microscopy, a laser scanning LSM 700 (Zeiss) was used.