Alexa fluor 647 conjugated donkey anti rabbit igg

Cross-sections of carotid arteries were used for confocal microscopic imaging. Platelets were stained with antibodies against CD42d (AF6990, R&D, USA), CD41 (553847, BD, USA) and PF4 (ab282111, Abcam, USA). Monocytes/macrophages were stained with rat anti-mouse F4/80 antibody (MAB5580, R&D, USA). Neutrophils were stained with Ly6G antibody (sc-52515, SANTA CRUZ, USA). Endothelial cells were stained with rabbit anti-mouse CD31 (ab28364, Abcam, USA). Podoplanin expression was determined with Syrian Hamster anti-mouse podoplanin antibody (14-5381-82, eBioscience, USA). Cytoskeletal proteins were determined with phalloidin antibody (CA1610, Solarbio, China). Secondary antibodies Alexa Fluor 488-conjugated donkey anti-rat IgG, Alexa Fluor 488-conjugated goat anti-mouse IgG, Alexa Fluor 647-conjugated donkey anti-rabbit IgG, and Alexa Fluor 647-conjugated goat anti-Syrian hamster IgG were all from Abcam. Donkey anti-sheep IgG NorthernLights™ NL557-conjugated antibody was purchased from R&D. Tissues were counterstained with DAPI before being mounted and examined by confocal microscopy, an Olympus multiphoton laser scanning microscope with high S/N ratio objectives and suppressed autofluorescence, which enables advanced deeper imaging with high resolution (FV1000MPE and FV3000MPE Olympus, Japan).

B2B cells were fixed with 4% paraformaldehyde, washed, and incubated with rabbit anti-human NF-кB p65 primary antibody (1 : 100 dilution; ABclonal, Woburn, MA, USA) overnight. Bound antibodies were detected with Alexa Fluor 647-conjugated donkey anti-rabbit IgG (1 : 200 dilution; Abcam, Cambridge, UK) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen). Fluorescence signals were captured using a fluorescence microscope (Olympus, Tokyo, Japan).