Goat anti rabbit af555

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.

For surface staining, cryostat sections (10μm thick) of lymph nodes were dried, fixed in 4% PFA for 10 minutes, blocked with PBS 5% BSA and then incubated with the following anti-mouse antibodies in 1% BSA for 1 hour: B220 Pacific Blue (RA3-6 B2, Biolegend), CD169 AF488 (Ser-4, ATCC), IgD FITC (11-26c.2a, BD Biosciences), Langerin AF647 (929F3.01, Dendritics) and GL-7 AF647 (GL7, Biolegend). For intracellular staining, sections were permeabilized with PBS Triton 0.3% for 5 minutes before blocking and then incubated with anti-mouse IL-1β (polyclonal, R&D), IL-18 (polyclonal, Abcam) or IL-33 (polyclonal, R&D), followed by incubation with AF555 anti-goat or anti-rabbit IgG (Invitrogen). CD1d tetramer staining was performed as previously described (Lee et al., 2015 (link)). Briefly, lymph nodes were incubated overnight with PE-labeled PBS-57 loaded CD1d tetramer in 2% FCS at 4°C. Next day, lymph nodes were washed and fixed with 4% PFA for 1 hour and snap frozen. 10μm sections were blocked with 5% BSA and stained with anti-PE antibody (polyclonal, Novus Biologicals) followed by goat anti-rabbit AF555 (Life Technology). Imaging was carried out on a LSM 780 (Zeiss) inverted confocal microscope using a Plan-Apochromat 40x NA 1.3 oil immersion objective.