Visualizing EgKI-1 and Tubulin in MDA-MB-231 Cells

MDA-MB-231 cells were grown in 8-well tissue culture chambers on slides (Sarstedt, Nümbrecht, Germany) overnight. On the following day, different chambers were treated with EgKI-1 (2 μM) and control buffer. After 24 hours incubation, supernatants were removed and cells were fixed in ice cold methanol for 10 min followed by two washes with PBS. Cells were then permeabilized with ethanol: acetic acid (2:1) at -20^°C for 5 min. After 3 × 1 min rinses in PBS, normal donkey serum (10%) was applied for 20 min at room temperature (RT). Excess normal serum was decanted and mouse anti-EgKI-1 antibody [16 (link)] and rabbit anti-tubulin antibody (Abcam, Cambridge, UK) diluted 1:80 in PBS were applied for 60 min at RT. Cells were washed with three changes of PBS and Alexa fluor donkey anti-mouse 488 and Alexa fluor donkey anti-rabbit 555 diluted 1:200 in PBS were applied for 30 min at RT. After washing with PBS, cells were stained with DAPI (1:35000 in PBS) for 2–5 min followed by another PBS wash. Prolong fluorescence mount (Thermo Fisher Scientific) was then applied to each slide and a coverslip applied. Cells were visualized under a Zeiss 780-NLO point scanning confocal microscope for the presence of EgKI-1 and tubulin. Fluorescence intensity of tubulin and total cell areas were then measured with ImageJ software [24 (link)] and statistically analyzed with GraphPad Prism version 7.