4.5. Immunofluorescence

The effects of parbendazole on cell microtubules in AsPC-1 and Capan-2 cells exposed to parbendazole, or to vehicle (DMSO) were examined by confocal microscopy. Briefly, 5 × 10³ cells were seeded in 8-well BD Falcon™ CultureSlides (BD Bioscience, San Jose, CA, USA) and incubated overnight at 37 °C, 5% CO₂, then treated with 0.2 µM, 0.7 µM parbendazole, or with vehicle control (DMSO) for 24 h. Cells were then fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min and washed with PBS. Following fixation, cells were incubated 60 min in 0.3% Triton™ X-100 (Sigma-Aldrich) in PBS+ (PBS, 5% FBS, 0.02% sodium azide and 10 mg/mL BSA), washed with PBS+ and then incubated with the anti-α-tubulin antibody (ab7291, Abcam, Cambridge MA, USA) in PBS+ overnight at room temperature. The primary antibody was detected using the Dylight conjugated anti IgG-heavy and light chain cross-adsorbed species-specific fluorescent secondary antibody (Bethyl Laboratories, Inc., Montgomery, TX, USA). Nuclear staining was performed with DRAQ5 (Cell Signaling Technology, Beverly, MA, USA). Coverslip were mounted with SlowFade^® Gold antifade reagent (Thermo Fisher Scientific, Life Technologies Italia Fil., Monza MB, Italia). All the images were acquired with identical settings and corrected by background subtraction using a Carl Zeiss LSM5 Pascal confocal laser scanning microscope (Carl Zeiss 40X Plan Neofluar oil-immersion objective, Carl Zeiss Microscopy, LLC, NY, USA).