4.14. Immunofluorescence Staining Analysis of Nuclear Translocation of NF-κB

The cells (1 × 10⁵ cells/mL) were cultured with DMEM medium, PSP-L (2000 μg/mL) or LPS (10 μg/mL) for 24 h. The culture medium was discarded, and fixing liquid was added to the cells. After 10 min, cells were washed three times with PBS for 5 min each washing. Next, cells were incubated with the primary NF-κB p65 antibody at 4 °C overnight and then washed with a washing solution three times for 10 min each, followed by incubation with a Cy3-conjugated secondary antibody at room temperature for another 1 h, and then washed two times. 4’,6-diamidino-2-phenylindole (DAPI) was added for staining and then removed after 5 min by washing three times. Finally, samples were visualized under a fluorescent inverted microscope (37XC, Shanghai optical instrument factory, Shanghai, China). All steps were under the instructions of NF-κB nuclear transfer kit and detection kit.