Anti vimentin monoclonal antibody

Huh-7, Bel-7402, SNU387, and SNU449 cells were seeded onto glass slides. At 48 h after transfection with the NAT10 siRNA or Twist siRNA or treatment with remodelin in the presence of doxorubicin or hypoxia, the cells were rinsed with PBS, fixed with 2% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked for 30 min in 10% BSA, and then incubated with an anti-E-cadherin monoclonal antibody (1 : 200; Cell Signaling Technology) or anti-vimentin monoclonal antibody (1 : 200; Cell Signaling Technology) overnight at 4°C. After three washes in PBS, the slides were incubated with goat anti-rabbit Cy3 as a secondary antibody (1 : 200; Cell Signaling Technology) for 1 h in the dark. After three further washes, the cells were stained with DAPI for 5 min to visualize nuclei and examined by confocal microscopy (Olympus, Tokyo, Japan).

Immunohistochemistry was done using a single-staining procedure. Anti-WFCD2, anti-Ecadherin,anti-Vimentin monoclonal antibody (Cell Signaling Technology), anti-CD44,anti-MMP2,anti-MMP9,and anti-ICAM-1 rabbit polyclonal antibody (Santa Cruz Biotechnology), were applied to the slides at a dilution of 1:1,00 ~ 1:150 in blocking buffer overnight at 4 °C. The slides were then washed and stained by the avidin-biotin method. The slides were lightly counter stained with hematoxylin. Tumor cells were considered positive for the antigen if there was brown color staining. The intensity was scored as negative (0), weak (1), medium (2), and strong (3),and the proportion of staining was scored as 1 (≤10%), 2 (11–50%), 3 (51–75%), and 4 (>75%). An overall expression score was calculated by multiplying the scores for intensity and proportion, ranging from 0 to 12. For ICAM-1, at least 500 tumor cell for each xenograft sample (n = 5) were randomly selected and counted. The number of positive cell was counted and the positive index was calculated as follows: ICAM-1 index = (number of stained cells/total cell number) × 100%.

Sections (4 μm) from each block were deparaffinized in xylene and rehydrated in a descending alcohol series. Antigen retrieval was performed by heating in a pressure cooker in 10 mmol/L citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by incubation in 0.3% H₂O₂ for 15 min, followed by incubation with 5% serum to reduce nonspecific binding. Sections were incubated with an anti-vimentin monoclonal antibody (1 : 1000; Cell Signaling Technology), anti-E-cadherin monoclonal antibody (1 : 1000; Cell Signaling Technology), or anti-Ki-67 monoclonal antibody (1 : 500; Cell Signaling Technology) at 4°C overnight. After washing in phosphate-buffered saline (PBS), slides were incubated with horseradish peroxidase-conjugated rabbit-anti-mouse secondary antibody, developed using 3,3-diaminobenzidine (DAB) chromogen solution and counterstained with Mayer's hematoxylin. Negative controls were performed in parallel by replacing the primary antibody with nonspecific serum.