Rabbit anti γ tubulin

Samples were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature, followed by three washes with PBS. To label E-cadherin, MKLP1, or Ki67, samples were blocked with 10% (vol/vol) goat serum (Sigma Aldrich) in PBS for 4 hr, and incubated overnight at 4°C with rabbit anti-E-cadherin antibody (Cell Signaling), rabbit anti-MKLP1 antibody (Abcam), or mouse anti-Ki67 antibody (Cell Signaling), respectively. Samples were washed 6 times with 0.3% Triton-X-100 in PBS (PBST) for 30 min each time, and incubated with Alexa 594 goat anti-rabbit secondary antibody (Invitrogen) overnight at 4°C. To label nuclei, samples were incubated with a 1:1000 dilution of Hoechst 33342 (Invitrogen) for 20 min at room temperature and washed 3 times with PBS for 10 min each time.
To label centrosomes, samples were fixed with 100% methanol for 20 min at −20°C, followed by three washes with PBS. Samples were blocked as described above and incubated with rabbit anti-γ-tubulin (Santa Cruz Biotechnology) overnight at 4°C. Samples were washed and incubated with secondary antibody as described above.
To assess proliferation rate in response to treatment with EGF, samples were incubated with 10 µM EdU for one hour before fixation, permeabilization, and EdU staining, using the Click-iT EdU Alexa Fluor 594 Imaging kit (Thermo Scientific) according to the manufacturer’s instructions.