Alexa fluor 594 labelled anti rabbit

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).

Paraffin-embedded tumor sections were incubated with primary antibodies against synaptophysin (clone SP11). The specific antibody binding was visualized using the Roche Ventana Ultra Immunostainer (DAB, Roche, Basel, Switzerland ). Hematoxylin-eosin was used as counterstaining. For immunofluorescence analysis, platelets of NEN patients and healthy donors were fixed in 4% PFA in PBS (10 min at 4 °C). Platelets were then blocked using a blocking solution containing 5% BSA, 0.2% Triton X-100, 0.1% Tween for 60 min. As primary antibody, we used anti-synaptophysin (1:200, Invitrogen, Carlsbad, CA, USA) and anti-CD41 (1:500, ThermoFisher, St. Louis, MO, USA); as secondary antibodies, Alexa-Fluor 594 labelled anti-rabbit (1:1000, Invitrogen, Carlsbad, CA, USA) 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).