Vinculin and Ki67 Immunofluorescence Assay

1 × 10⁴ cells
were seeded on the coverslips in the six-well plate and incubated
for 24 h and then treated with 10 mM metformin for 48 h. The coverslip
was fixed in 4% (v/v) paraformaldehyde for 15 min at room temperature
and permeabilized by 0.25% (v/v) Triton X-100 for 5 min. The coverslip
was blocked for 30 min in a blocking solution [1% (w/v) bovine serum
albumin (Bioshop) in PBS] at room temperature. Primary mouse anti-human
vinculin monoclonal antibody (1:200) (Millipore) and rabbit anti-human
ki67 polyclonal antibody (1:100) (Abcam, Cambridge, MA, USA) were
diluted in the blocking solution and incubated overnight at 4 °C.
After washing twice with PBST (PBS with 0.05% (v/v) Tween-20), samples
labeled with vinculin and ki67 were incubated in the secondary anti-mouse
IgG FITC antibody (1:100 in PBS; Invitrogen, Carlsbad, CA) and anti-rabbit
IgG FITC antibody (1:100 in PBS; Invitrogen, Carlsbad, CA), respectively,
with TRITC–phalloidin (1:1250 in PBS; Millipore) for 30 min
at room temperature, followed by washing three times with PBST. Subsequently,
coverslips were mounted with a ProLong Gold antifade reagent with
DAPI (Invitrogen) on the slide. Steps were described in our previous
paper.^{54 (link)}