Af2089

Cells were fixed with 4% paraformaldehyde in PBS, and permeabilised with 0.3% Triton-X detergent in PHEM buffer (10 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl₂, pH 6.9, all from Sigma-Aldrich). Primary antibodies against CD31 (1:50, JC70A, Dako, Glostrup, Denmark), LYVE-1 (1:100, AF2089, R&D Systems, MN, USA) CD32B (1:100, ab45143, Abcam, Cambridge, UK), Stabilin-2 (1:100, ab121893, Abcam), and Factor VIII (1:100, SAF8C-AP, Affinity Biologicals, ONT, Canada) were applied for 1 h, followed by secondary antibody for 30 min (either Alexa-Fluor 488 or 594 conjugated goat anti-mouse, goat anti-rabbit, or goat anti-sheep, all at 1:200, Thermo Fisher Scientific, MA, USA). After nuclear staining with DAPI, slides were mounted with fluorescence mounting medium (Dako) and a glass coverslip. Cells were imaged using a fluorescence microscope (Olympus BX61, Olympus, Tokyo, Japan).