Rabbit monoclonal antibody against vimentin

Total cell extracts and nuclear extracts were prepared using an extraction kit (Beyotime, Shanghai, China). 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels were used to separate 20 μg of cell lysates and the separated proteins were transferred to a nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Blots were probed with rabbit monoclonal antibody against Gal-1 (Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal antibody against vimentin (Cell Signaling Technology), rabbit monoclonal antibody against anti-E-cadherin (Cell Signaling Technology), or mouse monoclonal antibody against GAPDH (Kang Cheng, Shanghai, China) antibodies at a dilution of 1:2000. Horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin and HRP-conjugated goat anti-rabbit immunoglobulin were used as a secondary antibody at a dilution of 1:2000. The West Pico chemiluminescent Substrate (Pierce, Carlsbad, CA, USA) was used to visualize the immunoreactive protein bands, and densitometric image analysis software (Image Master VDS; Pharmacia Biotech) was used to quantify the visualized protein bands. The level of GAPDH was used as an internal reference. All experiments were performed in triplicate.

Tissues were fixed with 4% paraformaldehyde, dehydrated, embedded in paraffin and sectioned at 4 μm. Sections were deparaffinized, rehydrated and incubated with 3% H₂O₂. After antigen repair and being blocked, the slides were incubated with rabbit monoclonal antibody against vimentin (1:100) (Cell Signaling Technology, USA) at 4 °C overnight. Subsequently, the slides were incubated with secondary antibody at room temperature for 30 min and then incubated with streptavidin peroxidase complex. Staining was performed using 3, 3-diaminobenzidine (DAB) substrate kit for peroxidase reaction and counterstained with hematoxylin. Finally, the slides were analyzed with a light microscope.