Fluor 488 labeled anti mouse

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).

Tissue samples were fixed in 4% formalin and paraffin-embedded (FFPE) at the Department of Pathology (University Hospital Tuebingen). The sections were cut briefly in 3 µm sections and stained with Hematoxylin/Eosin and CD61 (clone: 2C9.G3) following standard protocols. For immunofluorescence microscopy, sections were deparaffinized and hydrated in a first step. The heat-induced antigen retrieval method was performed using sodium citrate buffer (pH 6.0) for 30 min. Antigen blocking was performed with Blocking solution (Zytomed) for 60 min. Primary antibodies that were included anti-CD41, mouse, 1:250 (clone: HIP8) and anti-PD-L1, rabbit, 1:200 (clone:28-8). Secondary antibodies include Alexa-Fluor 594 labeled anti-rabbit (1:1000, Invitrogen) and Fluor 488 labeled anti-mouse (1:1000, Invitrogen). DAPI (1:1000, BioLegend) was used for nuclear staining prior mounting the slides with H-1500 Vectashield Hardset. Microscopic analysis was done with an Olympus BX63 microscope and a DP80 camera (Olympus).