Rabbit monoclonal anti human vimentin

Protein contents of total cell lysates from TGF-β1 treated or untreated cells were analyzed by western blot. Samples with same amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, then the proteins were electro-transferred onto polyvinylidene difluoride membranes. The primary antibodies used were: mouse monoclonal anti-human GSN (Sigma GS-2C4; 1:10000 dilution), mouse monoclonal anti-human CD44 (Abcam;1:1000 dilution), mouse monoclonal anti-human E-cadherin (2Q663) (sc-71008), human-β-catenin (9 F2) (sc-47752), human GSK-3β (H-76) (sc-9166), human cyclin D1 (DSC-6) (sc-20044), mouse monoclonal anti-human N-cadherin (H-63) (sc-7939) (all from Santa Cruz; 1: 2000 dilution), mouse monoclonal anti-β-actin (Sigma, 1: 10000 dilution), rabbit monoclonal anti-Tm1 (Sigma, 1:2000 dilution), rabbit monoclonal anti-caldesmon (Santa Cruz, 1:5000 dilution), rabbit monoclonal anti-profilin (Santa Cruz, 1:3000 dilution), rabbit monoclonal anti-human vimentin (Abcam; 1:1000 dilution), and rabbit polyclonal anti-GAPDH (GeneTex GTX100118; 1:5000). The secondary antibodies used (1:5000 dilution) were goat anti-rabbit IgG (Sigma), and goat anti-mouse IgG (Sigma).

Primary EEC and EC cells (EM018a, EM033, EM012 and EM046) were seeded in a 12-well plate on collagen-coated (Sigma-Aldrich) coverslips (Karl Hecht). After EMT induction, cells were fixed with 4% formaldehyde for 10 min, permeabilized with 0.2% Triton X-100 for 10 min and blocked with 5% goat serum for 2 h. The primary antibodies were incubated overnight at 4 °C. The following primary antibodies were used: mouse monoclonal anti-human E-cadherin (1/30, Abcam), rabbit monoclonal anti-human vimentin (1/100, Abcam), mouse monoclonal anti-human cytokeratin (1/100 Agilent), mouse monoclonal anti-human MMP2 (1/100, Abcam), and mouse monoclonal anti-human α-smooth muscle actin (1/100, Agilent). The secondary antibodies (1:1000 in 0.5% goat serum, AlexaFluor488-conjugated anti-mouse IgG and anti-rabbit, AlexaFluor647-conjugated anti-mouse and anti-rabbit) were applied for 1 h at room temperature. Triple washing with PBS was performed between each step. Finally, the coverslips were mounted in medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vectashield, Vector Laboratories). Images were taken using the Nikon Fluorescence microscope (CIE i) taking care to use the same exposure and gain settings for each type of staining to compare fluorescence intensity.