Donkey anti rat igg h l alexa fluor 647

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).

Flow cytometry antibodies and reagents: FITC-conjugated anti-mouse CD11b, APC-conjugated anti-mouse CD45, PE-conjugated anti-mouse LAMP1 and PE-conjugated anti-mouse CD86 were purchased from Biolegend (USA), anti-CD16/CD32 Fc Block and myelin removal reads were purchased from Miltenyi Biotec (Germany). Western blot and immunofluorescence (IF) antibodies: IBA-1 (R&D systems, USA); VGLUT1 (Santa Cruz Biotechnology, USA); LAMP1 and CD68 were purchased from Abcam (United Kingdom); albumin, PSD95 and PKM2 were purchased from Proteintech (China). β-catenin, Cyclin-D1, c-Myc, ENO1, PFKFB3, HK1, LDHA, PDK1, GLUT1 were purchased from Cell Signaling Technology (USA), horseradish peroxidase (HRP)-linked goat anti-rabbit IgG (Fcmacs, China), NeuN (Santa Cruz Biotechnology), donkey anti-rat IgG H&L (Alexa Fluor® 647) (Abcam), goat anti-mouse IgG-TRITC (Abcam), rabbit anti-goat IgG-FITC (Fcmacs). ELISA kit: Maltose and Glucose assay kit (RayBiotech, USA), l-lactate assay kit (Cayman, USA), interleukin (IL)-6 (Fcmacs, China), IL-1β (Fcmacs, China). TRIzol reagent and SYBR green dye were bought from Invitrogen (Carlsbad, USA). PKM2 overexpression plasmid was purchased from Nanjing Jereh Company (China), and RFectPM Eukaryotic DNA Transfection Kit was purchased from Changzhou EMI Company (China). β-catenin protein inhibitor (KYA1797K) was purchased from MCE Company (USA).