Donkey anti rabbit igg h l alexa fluor 568

To evaluate the macrophage-platelet interaction during DBV infection, 8 × 10⁵ PMA-activated THP-1 cells were seeded onto a confocal plate (Biosharp, Anhui, China) and infected by DBV (MOI = 1). Macrophages treated with LPS and nigericin or untreated were served as positive and negative controls, respectively. After 72 h incubation, macrophages were washed three times with precooled PBS, fixed with 1% paraformaldehyde for 5 min, and penetrated with 0.1% Triton-X for 10 min. After blocking with immunostaining blocking solution (Beyotime, Shanghai, China) for 10 min, samples were incubated overnight at 4°C with anti-tubulin antibody (1:200) (Abcam, Britain), anti-caspase-1 antibody (1:200) (Santa Cruz Biotechnology, USA) and anti-DBV-Gc antibody (1: 200) (Sangon Biotech, Shanghai, China). Samples were washed with PBS and incubated with donkey anti-rabbit IgG H&L (Alexa Fluor 568) pre-adsorbed (1:200) (Abcam, Britain), goat anti-mouse IgG H&L (Alexa Fluor 488) pre-adsorbed (1:200) (Abcam, Britain) and donkey anti-rat IgG H&L (Alexa Fluor 647) pre-adsorbed (1:200) (Abcam, Britain) for 1 h. The nuclei were then stained using the mounting medium with DAPI (Abcam, Britain). The images were obtained using a laser confocal microscope (ZEISS, LSM880, Germany).