Cell proliferation was assessed with Ki-67 staining
After recovery, SKOV3 cells were washed with phosphate-buffered saline
(PBS) (Santa Cruz Biotechnology, Dallas, Texas, USA) and incubated in
4% paraformaldehyde (Santa Cruz Biotechnology, Dallas, Texas, USA) for
15 minutes. Cancer cells were washed with PBS plus 0.02% Triton X-100
(Sigma-Aldrich, Dorset, UK) 3 times for 5 minutes each and blocked with
10% donkey serum (Sigma-Aldrich, Dorset, UK) for 30 minutes. After
blocking, cells were incubated with anti-Ki-67 antibody (1:200, rabbit
polyclonal) (Santa Cruz Biotechnology, Dallas, Texas, USA) at 4°C
overnight and anti-rabbit antibody (1:1000, goat polyclonal) (Abcam,
Cambridge, UK) for 1 hour at room temperature under dark in sequence.
The 4’,6-diamidino-2-phenylindole (DAPI) mounting media (Vector
Laboratories, Burlingame, California, USA) were mounted on cancer cells.
The fluorescent images were taken under fluorescence microscope and the
percentage of Ki-67 positive cells was calculated by Fiji (ImageJ 2.0)
software (National Institutes of Health, Bethesda, Maryland, USA).