Eclipsete2000

Immunofluorescence was performed as described.^{33 (link)} The following primary antibodies and dilutions were used: rat anti-CD31 1 : 1000 (BD, Franklin Lakes, NJ, USA), mouse anti-Tuj-1 1 : 1000 (Covance, Princeton, NJ, USA), mouse anti-AFP 1 : 1000 (Inmunostep, Salamanca, Spain), rabbit anti-Nanog 1:1000 (Chemicon, Billerica, MA, USA), mouse anti-SSEA-1 (MC-480) 1 : 100 (Pierce, Waltham, MA, USA), rabbit anti-E-cadherin 1 : 60 (Cell Signaling, MA, USA), mouse anti-E-cadherin 1:200 (Cell Signaling, Danvers, MA, USA), rabbit anti-β-catenin 1 : 200 (BD). Secondary antibodies were: Alexa 647 goat anti-IgG rabbit (Molecular Probes, Eugene, OR, USA), Alexa 488 goat anti-IgG mouse (Molecular Probes), Alexa 568 donkey anti-IgG rat (Molecular Probes), Cy3 donkey anti-IgG rabbit (Jackson Immunoresearch, West Grove, PA, USA) and FITC donkey anti-IgG mouse (Jackson Immunoresearch). Images were obtained with NIKON EclipseTE2000 and ZEISS LSM 800 confocal microscope.

Cells were fixed for 20 min in 4% paraformaldehyde (Merck Life Science, Darmstadt, Germany) and blocked with 5% BSA (NZYTech) for 30 min. Cells were incubated with primary mouse antibody anti-E-cadherin (24E10) or anti-β-catenin (1:50; ON; 4 °C, Cell Signaling Technology, Danvers, MA, EUA) and primary rabbit monoclonal antibody anti-P-cadherin (#2130; 1:50; ON; 4 °C, Cell Signaling Technology), followed by secondary antibodies Alexa Fluor 488 Donkey Anti-Mouse (1:500; 60 min; Life Technologies) or Alexa Fluor 594 anti-rabbit (1:500, 60 min; Life Technologies). Cell nuclei were stained with DAPI (1 μg/mL in PBS; 5 min incubation; Sigma, Merck Life Science, Darmstadt, Germany). All coverslips were mounted using Vectashield mounting media (Vector Laboratories, Newark, CA, USA) and cells were analysed by fluorescence microscopy (Imager.Z1, AxioCam fluorescence microscope or Eclipse TE-2000, both from Zeiss, Oberkochen, Germany) using AxioVision software (Rockville, White Plains, NY, USA).