Mab278

Circular cover slips (12 mm diameter) were placed in wells of a 24-well tissue culture plate and coated with recombinant human fibronectin for 2 h at 37°C or overnight at 4°C. Cells were grown on these cover slips for 18–20 h in complete vascular cell growth medium at 37°C with 5% CO₂. Samples were fixed with paraformaldehyde (4%, Thermo Scientific)) in PBS and 2% sucrose for 30 min at RT. For permeabilization, samples were incubated with 0.1% Triton X-100 for 5 min and after extensive washing, blocked with 2% BSA/PBS for 30 min at RT. Samples were incubated with primary antibodies against chemokines CCL2 (5 μg, MAB679) CCL5 (5 μg, MAB278), CCL7 (5 μg, MAB282), CCL8 (5 μg, MAB281), CCL20 (5μg, AF360), CXCL9 (5 μg, MAB392), mouse IgG₁ isotype control (5 μg, MAB002) and mouse IgG_2B isotype control (MAB004) all from R&D Systems, at RT for 2 h. Cells were washed three times with PBS before incubating with secondary antibody Alexa Fluor 488 (1:500, A-11017; Invitrogen) for 1 h and after washing three times with PBS, were counter-stained with DAPI (1μg/ml 62248; Invitrogen). Images were captured using a Leica SP8 (690) fluorescence microscope. Images were obtained with 40x objective. Images were analyzed using IMARIS software (Oxford Instruments).