Anti cd61

For immunofluorescence staining, platelets and Meg-01 cells were fixed in 4% PFA in PBS (10 min at 4 °C). Platelets and cells were blocked using a BSA blocking solution containing 5% BSA, 0.2% Triton X-100, 0.1% Tween for 60 min. As primary antibody we used anti-GITRL (1:200, R&D Systems) and anti-CD61 (1:500, ThermoFisher, St. Louis, MO); as secondary antibodies Alexa-Fluor 594 labeled anti-rabbit (1:1000, Invitrogen, Carlsbad, CA) and Fluor 488 labeled anti-mouse (1:1000, Invitrogen) were used. Slides were mounted in fluorescent mounting medium; for Meg-01 cells 0.5 μg/ml Hoechst was used for counter-staining. Plasma membranes were stained using Dil (ThermoFisher), nuclear staining was done via NucBlue™ (ThermoFisher) according to manufacturer’s instructions. Pictures were acquired using an Olympus BX63 microscope and a DP80 camera (Olympus, Shinjuku, Japan).

For immunofluorescence analysis, platelets of breast cancer patients and healthy donors (HD) were fixed in 4% PFA in PBS (10 min at 4°C). Platelets were blocked using a BSA blocking solution containing 5% BSA, 0.2% Triton X-100, 0.1% Tween for 60 minutes. As primary antibody anti-TACI (1:200, R&D Minneapolis, MN) and anti-CD61 (1:500, ThermoFisher, St. Louis, MO) were used; as secondary antibodies Alexa-Fluor 594 labelled anti-rabbit (1:1000, Invitrogen, Carlsbad, CA) and Fluor 488 labelled anti-mouse (1:1000, Invitrogen) were used. Slides were mounted in fluorescent mounting medium. Pictures were acquired using an Olympus BX63 microscope and a DP80 camera (Olympus, Shinjuku, Japan). Quantification of platelet size and fluorescence intensity was performed using an ImageJ script (v.1.52).