Zeis lsm 5 exciter confocal laser scanning microscope

Cells were grown on poly-L-Lysine-treated coverslips and after 6h of the different treatments the culture media was discarded, cells were washed thrice in PBS and permeabilized in methanol. After washing with PBS, coverslips were incubated with Anti-b-Catenin (1/250) mouse mAb (BD) and FOXM1 (1/250) rabbit pAb (Santa Cruz Biotechnology), for 1h at room temperature. Coverslips where washed thrice in PBS and incubated for 1h at room temperature with an anti-mouse IgG alexa fluor 488-labeled antibody (1/500) (Molecular Probes, Eugene, OR, USA) and with anti-rabbit IgG (H+L) alexa fluor 594-labeled antibody (1/500) (Santa Cruz Biotechnology). Finally, cells were incubated with 300 nM 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) in PBS for 5 minutes at room temperature. Coverslips were mounted using Aqua Poly/Mount (Polysciences, Warrington, PA, USA) and visualized in a Zeis LSM 5 Exciter Confocal laser-scanning microscope (Zeiss, Germany). Images were analyzed using the image-J software (National Institutes of Health).

Cells were grown on poly-L-Lysine-treated coverslips to form a nearly confluent monolayer and after 6h of the different treatments. Then, the culture media was discarded, cells were washed thrice in PBS and permeabilized in methanol. After washing with PBS, coverslips were incubated with Anti-β-Catenin (1/250) mouse mAb (BD) and FOXM1 (1/250) rabbit pAb (Santa Cruz Biotechnology), for 1h at room temperature, washed thrice in PBS and labeled with an anti-mouse IgG alexa fluor 488-labeled antibody (1/500) (Molecular Probes, Eugene, OR, USA) and with anti-rabbit IgG (H+L) alexa fluor 594-labeled antibody (1/500) (Santa Cruz Biotechnology), for 1h at room temperature. Finally, cells were incubated with 300 nM 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) in PBS for 5 minutes at room temperature. Coverslip were mounted using Aqua Poly/Mount (Polysciences, Warrington, PA, USA) and visualized in a Zeis LSM 5 Exciter Confocal laser-scanning microscope (Zeiss, Germany). Images were analyzed using the image-J software (National Institutes of Health).