Rabbit polyclonal anti vimentin

To demonstrate protein co-localizations and to confirm protein-protein interactions predicted by STRING analysis, immunofluorescence study was performed. MDCK cells were cultivated on a cover slip for 24 h and the cells were fixed with 3.7% formaldehyde/PBS for 15 min and permeabilized with 0.1% triton X-100/PBS for 15 min. The cells were then incubated with mouse monoclonal anti-α-tubulin, rabbit polyclonal anti-vimentin, or goat-polyclonal anti-ANXA2 (all were purchased from Santa Cruz Biotechnology Inc. and were diluted 1:50 in 1% BSA/PBS) at 37 °C for 1 h. After washing, the cells were incubated with corresponding secondary antibody conjugated with Alexa 488 (in green; for α-tubulin) or Alexa 555 (in red; for vimentin and ANXA2) (Invitrogen-Molecular Probes; Burlinton, ON, Canada; all were diluted 1:500 in 1% BSA/PBS) at 37 °C for 1 h, whereas 0.1 μg/ml Hoechst dye (Invitrogen-Molecular Probes) was also co-incubated for nuclear staining. The cells were finally mounted with 50% glycerol/PBA and images were captured under an ECLIPSE 80i fluorescence microscope (Nikon; Tokyo, Japan).

Antibodies from the indicated manufacturers used in this study were as follows: mouse monoclonal anti-CK8 (1/200 dilution), rabbit polyclonal anti-CK14 (1/200 dilution) (Covance, CA); rabbit polyclonal anti-AR (1/50 dilution) and rabbit polyclonal anti-Vimentin (1/50 dilution) (Santa Cruz Biotechnology, CA); Rabbit polyclonal anti-beta III Tubulin antibody (1/50 dilution) (Abcam Inc., MA); rhodamine phalloidin (1/100 dilution); Alexa 488 goat anti-mouse, goat anti-rabbit, Alexa 568 goat anti-mouse, goat anti-rabbit (Invitrogen, CA); Biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA). All secondary Alexa Fluor antibodies were used at 1/100 dilution, and the secondary biotinylated antibody was used at 1/200 dilution. Fluoro-gel II with Dapi was purchased from EMS (Electron Microscopy Sciences, PA).