Fluoroview software

Ibidi 8 well µ-slides (Ibidi cat# 80826) were coated with 0.01% poly-L-lysine. SW480 cells were plated onto the slides and cultured for 3 days or in case of synchronization, 2 days in complete medium before they were synchronized by a double thymidine block. Within an hour after release from the second block, vehicle or drugs were added at the stated final concentrations (0.1 or 1.0 μM). Cells were incubated with the drugs for 24 h, then fixed in 4% formaldehyde/PBS for 10 min at room temperature (RT), permeabilized with 0.2% Triton X-100 in PBS for 5 min at RT and incubated in PBS containing 0.2% Bovine Serum Albumin for 1 h at RT to block non-specific binding of antibodies. Cells were stained with the β-tubulin primary antibody (1 h at RT, 1:500), washed and incubated with AlexaFluor488-labeled secondary antibody (1:500) and Phalloidin (1:1000) for 1 h at RT, followed by a 5-min incubation with DAPI (0.1 µg/ml). Immunofluorescent staining of the cells was detected with an Olympus FV1000 Spectral Confocal attachment to an Olympus IX-81 microscope on a 60x water immersion lens. Images were collected using standard filter sets and laser lines (405 nm, 488 nm and 594 nm). The images were captured using Olympus FluoroView software (Version 1.7c). The images were imported into the ImageJ/Fiji^{49 (link)} software for processing and visualization using Bioformats^{50 (link)} plugins.