Alexa fluor 488 conjugated donkey anti rabbit igg

IFAs were performed to examine the subcellular localization of endogenous IRF3 and NF-κB subunit p65 in LLC-PK1 or HEK-293T cells. Cells seeded on microscope coverslips in 24-well plates were transfected with the indicated expression plasmid when the cells reached approximately 80% confluence. After 24 h, the cells were mock-infected or infected with SEV for 6 h. The cells were fixed with 4% paraformaldehyde for 15 min and then permeated with methyl alcohol for 10 min at room temperature. After three washes with PBST, the cells were sealed with PBST containing 5% bovine serum albumin (BSA) for 1 h, followed by incubation separately with a rabbit polyclonal antibody against IRF3 (1:200) or against p65 (1:200) or a mouse anti-HA antibody (1:200) for 1 h at room temperature. The cells were then treated with secondary antibodies Alexa Fluor 594-conjugated donkey anti-mouse IgG and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Santa Cruz Biotechnology) for 1 h at 37 °C, followed by treatment with 4ʹ, 6-diamidino-2-phenylindole (DAPI) (Beyotime, China) for 15 min at room temperature. After the samples were washed with PBST, fluorescent images were visualized and examined with a confocal laser scanning microscope (Fluoviewver. 3.1; Olympus, Japan).