Alexa fluor 488 labeled goat anti rabbit igg

HepG2 cells treated with or without berberine were fixed in 4.0% polyformaldehyde for 15 min, washed with PBS, and then treated with permeabilization solution (PBS, 0.25% Triton X-100) for 10 min. After the cells were washed with PBS, they were blocked with 3% bovine serum albumin in PBS for 1 h at room temperature. Cells were stained with anti-caspase 8 antibody or anti-NF-κB antibody overnight at 4°C, followed by incubation with an Alexa Fluor^® 488-labeled goat anti-rabbit IgG (Cell Signaling Technology) antibody at room temperature for 1 h. Cells were then incubated with 2 μg/ml of DAPI (Invitrogen) in PBS for 5 min. Coverslips were mounted, and images were acquired with fluorescence microscopy (Olympus IX71). Alexa Fluor1 pt 488 and DAPI images were taken from the same field.

Berberine and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-NF-κB p65 and rabbit anti-caspase 8 antibodies were purchased from Abcam (Cambridge, MA, USA). Rabbit anti-GAPDH antibody and Alexa Fluor^® 488-labeled goat anti-rabbit IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). All other chemicals were of analytical grade unless indicated otherwise. The NF-κB p65 expression vector pCMV4-p65 (plasmid #21966; Addgene) and empty vector (pCMV4-3HA; plasmid #24165; Addgene) were a gift from Dr. Warner Greene.