Anti vimentin monoclonal antibody

FHUS were fixed in 4% paraformaldehyde for 30 min at 25°C. Following fixation, cells were permeabilized with 0.3% (v/v) Triton X-100 in PBS and then blocked with 5% bovine serum albumin. Next, the cells were incubated with the anti-vimentin monoclonal antibody (1 : 100; cat number ab8978; Abcam, Cambridge, UK) at 37°C for 1 h and then incubated with the secondary antibody for 30 min at room temperature (1/1000; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China). The slides were examined using a Nikon DS-Ri1 Eclipse microscope (Nikon, Tokyo, Japan).

CE81^FN+, CE81^FN−, CE81^FN+ + CON and CE81^FN+ + 146a cells were seeded (4 × 10⁴ cells/well) onto glass coverslips in a 6-well plate. The cells were fixed in 3.7% paraformaldehyde for 30 min, and washed by 1X PBS for 5 min followed by treatment with 0.1% Triton-X-100 for 30 min. Cells were blocked in 1X blocking buffer for 1 h at RT, and then treated with the monoclonal anti-vimentin antibody (Abcam, Cambridge, UK) for 16 h at 4 °C. After PBS washing for 10 min, cells were treated with secondary mouse monoclonal antibody and kept in the dark for 1 h. After PBS washing, cells were counterstained with Hoechst 33342 staining solution (Abcam) for 30 min and then mounted with glycerol gelatin (Sigma). The protein level of vimentin was evaluated by IHC staining of tumor and non-tumor tissues of the ESCC patient specimens. The anti-vimentin monoclonal antibody (Abcam) was used to detect vimentin protein. The secondary antibody was used followed by Streptavidin labeling (Dako, Cytomation, Carpinteria, USA). The slides were then treated with AEC solution for 20 min at RT, and counterstained with 10% hematoxylin (Muto Pure Chemicals, Tokyo, Japan) and mounted with glycerol gelatin (Sigma).

Cell culture reagents were from Invitrogen Life Technologies (Thermofisher). Anti-HNE-Michael adduct antibodies were from Oxis Research (#24327) for immunofluorescence studies, and from Invitrogen (#MA5-27570), for immunoprecipitation experiments. The anti-vimentin monoclonal antibody was from Abcam (#ab92547). Anti-γH2AX (#9718S), anti-SIRT1 (#9475S), anti-acetylated-Lysine (#9441S), and secondary anti-mouse and anti-rabbit HRP-conjugated antibodies were from Cell Signaling Technology. Anti-ubiquitin antibody was from Santa Cruz Biotechnology (#sc-8017). 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), L-Carnosine, 2-phenylindole dihydrochloride (DAPI), and anti β-actin antibody were from Sigma-Aldrich. Secondary Alexa Fluor antibodies 488 and 546 were from Life Technologies.