Immunofluorescence Analysis of Cell Markers

Cells were seeded on glass coverslips, fixed with 4% PFA (Sigma-Aldrich) and incubated overnight at 4°C with the following primary antibodies: anti-PanCytokeratin (Mouse 1:200, Dako, Glostrup, Denmark), anti-Epcam (Mouse 1:1000, clone HEA-125, GeneTex, Irvine, CA, USA), anti-AQ-1 (Mouse 1:50, clone B-11, Santa Cruz Biotechnology, Heidelberg, Germany), anti-CD13-PE (Mouse 1:25, Biolegend, San Diego, CA, USA), anti-CD13-FITC (Mouse 1:25, Abcam, Cambridge, UK), anti-N-cadherin (Rabbit 1:50, Abcam, and Mouse 1:50, clone 32/N, Becton Dickinson, San Josè, CA, USA), anti-Calbindin (Mouse 1:100, clone CB-955, Sigma-Aldrich), anti-E-cadherin (Mouse 1:50, Becton Dickinson, and Rabbit 1:50, Cell Signalling Technology, Danvers, MA, USA) and anti-Paxillin (Mouse 1:50, Becton Dickinson). When necessary, the secondary antibodies Alexa 488 conjugated anti-mouse IgG and Alexa 594 conjugated anti-rabbit IgG (1:100, Molecular Probes, Carlsberg, CA, USA) were used. Stress fibers were labeled by Alexa-Fluor-594-phalloidin (1:100, Molecular Probes) and nuclei counterstained with Mounting DAPI (Molecular Probes). Immunofluorescence images were obtained with a Zeiss LSM810 confocal microscope, using a 63x objective, equipped with Zen2009 software (Zeiss, Oberkochen, Germany).