C3d rabbit polyclonal antibody

Platelets, megakaryocytes, HepG2 cells and HL‐60 cells were lysed in RIPA buffer. After determination of the protein concentration by bicinchoninic acid analysis (Thermo Scientific), 18 μg of total protein was mixed with 100 mm dithiothreitol and Nupage loading buffer (Invitrogen), and boiled for 5 min. The presence of complement proteins was tested for with reducing SDS‐PAGE on 6–7% polyacrylamide gels under standard conditions. Proteins were transferred to nitrocellulose membranes, which were blocked with 5% skimmed milk in PBS containing 0.1% Tween‐20 prior to an overnight incubation with C3c–horseradish peroxidase (HRP) sheep polyclonal antibody (1 : 1000; Life Span Biosciences, Seattle, WA, USA) or C3d rabbit polyclonal antibody (1 : 3000; Dako). Goat anti‐rabbit H + L HRP (1 : 50 000; BioRad, Hercules, CA, USA) was used as the secondary antibody, and the signal was developed with the ECL West Pico and West Femto detection system (Thermo Scientific).

For immunocytochemistry, cytospins of megakaryocytes were prepared according to standard procedures. They were stained with C3c chicken polyclonal antibody (1 : 300; Antibodies Online, Aachen, Germany), C3d rabbit polyclonal antibody (1 : 150, Dako), anti‐rabbit APC (Invitrogen, Life Technologies, Carlsbad, CA, USA), and CD61–FITC (1 : 200; eBioscience), or isotypes in 3% PBS 0.1% BSA and Triton X‐100 for 1 h at room temperature. After being washed in PBS, the slides were stained with secondary donkey anti‐chicken Cy5 antibody (1 : 500; Jackson Immuno Research, West Grove, PA, USA) or goat anti‐rabbit AF555 (1 : 2000; Invitrogen) for 45 min at room temperature. Platelets from PCs or resting platelets were attached to polylysine‐coated glass slides in the presence of 1 μm PGI₂ (Sigma‐Aldrich), before being stained with C3c, C3d and CD61 antibodies. An LSM 700 confocal microscope (Carl Zeiss, Jena, Germany) with a × 63 objective and zen 2009 black software were used for the immunocytochemical analysis.