2.10. Immunofluorescence assayAfter

2.10. Immunofluorescence assay
After exposure to segetoside I (0, 0.82, 8.2, 82.0 M) or 5-FU
(0, 9.23, 92.3, 923 M), HepG2 cells were fixed with fresh 4%
paraformaldehyde at 4 ◦C overnight and washed (3 x) with PBS. The
cells were incubated with 0.3% Triton X-100 solution for 30 min to
increase the permeability of the cytomembrane. Then, 3% bovine
serum albumin (BSA) was added, followed by incubation for 1 h
at room temperature to block nonspecific binding sites. The cells
were further incubated at 4 ◦C overnight with an anti-Bax (1:500)
or anti-Bcl-2 (1:500) primary antibody. It was then washed and
incubated at room temperature for 4 h with a goat anti-mouse
Cy3-conjugated or goat anti-rabbit Cy3-conjugated secondary antibody
(1:500) and counterstained with DAPI. The cells were finally washed as described previously and observed under fluorescence microscope.