Alexa fluor 594 conjugated goat anti rabbit igg secondary antibody

RAW 264.7 cells were inoculated to 6 well cell culture plates, and the next day the medium was replaced with material leach liquors containing LPS (1 µg mL⁻¹) for 24 h. The cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized by 0.5% Triton X‐100 for 15 min. After three washes with PBS, cells were blocked using 10% normal goat serum. Subsequently, cells were incubated with anti‐CD206 (host: rabbit) and anti‐iNOS (host: mouse) primary antibodies (1:500; Abcam, Cambridge, UK) at 4 °C overnight, washed with PBS and incubated with Alexa Fluor 594‐conjugated goat anti‐rabbit IgG secondary antibody and Alexa Fluor 488‐conjugated goat anti‐mouse IgG secondary antibody (1:500; Proteintech, Wuhan, China) in the dark for 1 h at 37 °C. Nuclei were redyed using 2‐(4‐Amidinophenyl)−6‐indolecarba midine dihydrochloride (DAPI; Solarbio). Images were observed under a fluorescence microscope (Leica DMi8, Wetzlar, Germany) in the darkroom.

RAW264.7 cells plated in 24-wells plate were fixed with 4% paraformaldehyde for 10 min in a fume hood, permeabilized using 0.5% Triton X-100 (Solarbio, Beijing, China) for 10 min and rinsed three times with cold PBS for 5 min for each. After being blocked with 5% goat serum for 1 h, cells were incubated with an anti-NF-κB p65 primary antibody (1:500; CST) overnight at 4 °C. After washed with cold PBS twice, 1‰ Tween PBS once and incubated with Alexa Fluor 594-conjugated goat anti-rabbit IgG secondary antibody (1:500; Proteintech) for 1 h in the dark, nuclei were stained with DAPI (Proteintech). Images were detected with fluorescence microscope (OLYMPUS, Tokyo, Japan).