Fitc conjugated anti f actin

After the specific time of incubation, DU145 and DU145R80 cells were fixed in p-formaldehyde (4% v/v in PBS) for 5 minutes. The cells were permeabilized in Triton X-100 (0.5% v/v in PBS) for 5 minutes, and then incubated in goat or donkey serum (20% v/v PBS) for 30 minutes, and with primary antibodies against ANXA1 (rabbit polyclonal; 1:100; Invitrogen), vimentin (mouse monoclonal; 1:500; Santa Cruz Biotechnology) and FAK (mouse monoclonal; 1:100; BD Transduction Laboratories), overnight at 4°C. After two washing steps with PBS, cells were incubated with anti-rabbit and / or anti-mouse AlexaFluor (488 and/or 555; 1:1000; Molecular Probes) for 2 hours at RT and then with FITC-conjugated anti-F-actin (5 μg/ml; Phalloidin-FITC, Sigma) for 30 minutes at RT in the dark. Hoechst 33342 (Molecular Probes) was used to detect nuclei. The coverslips were mounted in Mowiol (Mowiol 4–88, Sigma-Aldrich). A Zeiss LSM 710 Laser Scanning Microscope (Carl Zeiss MicroImaging GmbH) was used for data acquisition. Images were generated with Zeiss ZEN Confocal Software (Carl Zeiss MicroImaging GmbH).

After the specific time of incubation, MIA PaCa-2, PANC-1, BxPC-3 and CAPAN-2 cells were fixed in p-formaldehyde (4% v/v in PBS) for 5 minutes. The cells were permeabilized in Triton X-100 (0.5% v/v in PBS) for 5 minutes, and then incubated in goat serum (20% v/v PBS) for 30 minutes, and with rabbit anti-ANXA1 antibody (1:100; Invitrogen), mouse anti-FAK (1:100; BD Transduction Laboratories), mouse anti-E-cadherin (1:250; Santa Cruz Biotechnology) and/or mouse anti-vimentin (1:500; Santa Cruz Biotechnology) overnight at 4°C. After two washing steps with PBS, cells were incubated with anti-rabbit and/or anti-mouse AlexaFluor (488 and/or 555; 1:1000; Molecular Probes) for 2 hours at RT and then with FITC-conjugated anti-F-actin (5 μg/ml; Phalloidin-FITC, Sigma) for 30 minutes at RT in the dark. The coverslips were mounted in glycerol (40% v/v PBS). A Zeiss LSM 710 Laser Scanning Microscope (Carl Zeiss MicroImaging GmbH) was used for data acquisition. To detect nucleus, samples were excited with a 458 nm Ar laser. A 555 nm He-Ne laser was used to detect emission signals from ANXA1 stain. Samples were vertically scanned from the bottom of the coverslip with a total depth of 5 mm and a 63× (1.40 NA) Plan-Apochromat oil-immersion objective. Images were generated with Zeiss ZEN Confocal Software (Carl Zeiss MicroImaging GmbH).