Rabbit anti anxa1 antibody

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).