8 well glass bottom slides

For immunofluorescent staining of SCARF-1 in HSEC, cells were cultured to confluence on rat tail collagen (RTC)-coated 8-well glass bottom µ-slides (Ibidi^®), subsequently stimulated (see ‘HSEC stimulation’) and fixed in 4% paraformaldehyde (PFA). Alternatively, HSEC were cultured overnight in RTC-coated μ-Slides VI 0.4 (Ibidi^®), stimulated for a further 24 h with 10 ng/ml TNFα and had 1 × 10⁶ cells/ml CD4⁺ T lymphocytes perfused over them as described below (see ‘Flow adhesion assays’). HSEC and adherent CD4⁺ T cells were then fixed in 4% PFA. Following fixation, all cells were washed in PBS, permeabilised with PBS with 0.3% Tween^® 20 for 5 min and then blocked in PBS with 10% goat serum for 20 min. The cells were then incubated at room temperature with anti-SCARF-1 (12 μg/ml; Abcam; ab92308) and anti-ICAM-1 (10 μg/ml; R & D; BBA3) primary antibodies, Alexa Fluor™ 633 Phalloidin (1 in 40; Invitrogen) or IMC antibodies diluted in PBS for 1 h. The cells were then washed with PBS three times and incubated with appropriate Alexa Fluor^® conjugated secondary antibodies (1:250; Thermo Fisher Scientific). Nuclei were labeled with 300 nM DAPI. Cells were washed with PBS three times and left in PBS after final wash before imaging on a Zeiss 780 Zen confocal fluorescence microscope (ZEISS).