Anti a actin n 19

For immunofluorescence assay, cells were seeded onto multi-chamber slides (Becton–Dickinson) at a density of 1  ×  10⁴ cells. After 5 days of treatment, the slides were washed in phosphate-buffered saline (PBS) with calcium and magnesium, fixed in paraformaldehyde 4% for 15 min and permeabilized in Tris buffered saline (TBS) 0.05 M pH 7.4 with 1% Triton X-100 for 15 min. After blocking in TBS 10% serum for 30 min, slides were incubated with the monoclonal antibodies: anti-Human N-cadherin (1 mg/mL) (R&D system AF 6426), anti-vimentin cloneV9 (1:250) (SIGMA), anti-a-actin (N-19) (1:200) (Santa Cruz), anti E-cadherin (G-10) (Santa Cruz) and anti-cytokeratin, pan (mixture) C2562 (SIGMA), for 1 h at 4 °C. After washing in TBS, they were incubated with goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 594 antibodies (invitrogen) for 30 min and finally washed twice in TBS. Nuclei were counterstained with Hoechst 33342 (Sigma) 1 μg/mL for 10 min. Images were acquired with an Olympus BX51 fluorescence microscope and analyzed with I.A.S. software (Delta Sistemi, Legnano, Italy). The brightness and contrast of the acquired images were adjusted, and the figures were generated using Adobe Photoshop 7.0. Cells not treated with RA were considered to be negative controls.

Proteins extracted from fibroblast homogenates were separated by 10% SDS–polyacrylamide gel electrophoresis and then transferred to a 0.45 μm pure nitrocellulose membrane (Bio-Rad) by transfer buffer. The blots were blocked with milk at 5% for 1 h and incubated overnight at 4 °C with the monoclonal antibodies: anti-Human N-cadherin (1 mg/mL) (R&D system), anti-vimentin cloneV9 (1:250) (SIGMA), anti-a-actin (N-19) (1:200) (Santa Cruz), anti E-cadherin (G-10) (Santa Cruz) and anti-cytokeratin, pan (mixture) C2562 (SIGMA), anti-β-catenin (E-5) (Santa Cruz) and anti-MMP-9 (626–644) (Ab-3) (Calbiochem). Primary antibodies-bound membranes were incubated at 4 °C overnight with HRP-conjugated secondary antibody (1:4000) anti-mouse (SIGMA). Further details are reported in a previous study [19 (link)]. For detection, an ECL chemiluminescence system (Therma Scientific) was used.