Epr1942

Isolated monocytes containing platelets were spotted on Alcian blue‐coated glass slides and fixed in cold 4% paraformaldehyde (4°C, 30 min). After fixation, cells were washed with PBS and then blocked in PBS with 0.2% BSA (for surface staining) or PBS with 0.1% Triton and 0.2% BSA (T‐PBS; for intracellular staining) for 30 min at room temperature shaking. Following blocking, monocytes and platelets were incubated with primary specific antibodies against Annexin A1 (AnxA1; 5 μg/ml; clone 1B, in house generated) and Gelsolin (GSN; 1.54 μg/ml clone EPR1942; Abcam, Cambridge, UK; Cat#ab109014, RRID:AB_10863643) in either PBS+0.2% BSA or T‐PBS+0.2% BSA overnight at 4°C. Cells were washed and incubated with secondary antibody Alexa Fluor 488 anti‐rabbit (5 μg/ml, Molecular Probes Invitrogen, Eugene, USA; Cat#A‐11008, RRID:AB_143165) or Alexa Fluor 594 anti‐mouse (5 μg/ml, Molecular Probes Invitrogen; Cat# A‐11032, RRID:AB_2534091) in T‐PBS+0.2% BSA for 1 h at 20°C shaking. Cells were then mounted with a glass coverslip using Fluoroshield Histology Mounting Medium with DAPI (Sigma‐Aldrich) and visualized under the microscope Zeiss LSM800 Imaging System.