Evos sl digital inverted microscope

CCRF-CEM cells were treated with 30 µM of compound (1) for 24 h. Then, the cells were washed with PBS and fixed with 3.7% paraformaldehyde for 30 min at room temperature. The cells were blocked for 1 h at room temperature using a blocking buffer (5% FBS and 0.3% Triton X-100 in PBS). Primary antibodies [rabbit α-tubulin antibody (Abcam, Cambridge, UK) and mouse PLK1 monoclonal antibody (Thermo Fisher Scientific)] were added and allowed to stand for 2 h at room temperature (dilution 1:1000). Then, the cells were washed with PBS three times. Secondary antibodies [goat anti-rabbit IgG H&L (Alexa Fluor^® 488) (Abcam) or goat anti-mouse IgG H&L (Cy3^®) preadsorbed (Abcam)] were subsequently added (dilution 1:1000). Then, the cells were washed with PBS, stained using 1 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) (Sigma Aldrich), and mounted using Fluoromount-G^® (Southern Biotech, Birmingham, AL, USA). Fluorescence imaging was performed using an EVOS SL digital inverted microscope (Life Technologies).

A549 cells treated with 40 µmol/L of MCC1019 for 24 h were washed with PBS and fixed using 3.7% paraformaldehyde at room temperature for 30 min. After blocking with 5% FBS and 0.3% Triton X-100 in PBS for 1 h at room temperature, rabbit α-tubulin antibody (ab52866, Abcam) was added for 2 h at room temperature. Secondary antibody goat anti-rabbit IgG H&L (Alexa Fluor^® 488) was added for 1 h at room temperature. Wash steps after each antibody incubation were performed in triplicate with PBS. Cells were additionally stained using 2 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) (Sigma–Aldrich), and mounted in Fluoromount-G^® (SouthernBiotech, Birmingham, AL, USA). An EVOS SL digital inverted microscope (Life Technologies) was used for fluorescent imaging.