Anti vimentin rabbit monoclonal antibody

PC9 cells were fixed in 4 wt% paraformaldehyde (Sigma, St Louis, MO, USA) for 30 min and rinsed three times with PBS for 10 min each time. The samples were immersed in 0.2% Triton X-100 for 10 min, rinsed three times for 10 min each time with PBS, and then blocked in 4% goat serum for 1 h at room temperature. Then sample sections were incubated overnight in anti-E-cadherin rabbit monoclonal antibody (1:200, Cell Signaling Technology) and anti-vimentin rabbit monoclonal antibody (1:200, Cell Signaling Technology) at 4°C. After rinsed with PBS three times, the samples were subsequently incubated in anti-rabbit immunoglobulin G secondary antibody conjugated with fluorescein isothiocyanate (FITC) or APC (1:100, eBioscience) for 1 h in the dark. For nuclei observation, the samples were dipped in 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) solution (Sigma, 3 μg/mL) and immediately rinsed with PBS. In the stained image, the E-cadherin displayed red fluorescence, the vimentin displayed green florescence, and the nuclei displayed blue fluorescence.

Formalin-fixed, paraffin-embedded tumor tissue was cut into 4-μm thick sections and immunohistochemistry was performed using a protocol previously reported by our group but with some modifications[15 (link)]. The most representative section of tumor for each case was selected for analysis. We analyzed not only DCLK1 expression, but also the expression of E-cadherin, N-cadherin, vimentin, and Snail as EMT markers, and CD24, CD44, CD133, and epithelial cell adhesion molecule (EpCAM) as CSC markers. The primary antibodies used for immunohistochemistry were: rabbit polyclonal anti-DCLK1 antibody (1:80 dilution; Abcam, Cambridge, MA, United States); rabbit polyclonal anti-Snail antibody (1:80 dilution; Abcam); mouse monoclonal anti-E-cadherin antibody (1:50 dilution; Dako Co., Carpinteria, CA, United States); rabbit monoclonal anti-vimentin antibody (1:100 dilution; Cell Signaling, Danvers, MA, United States); rabbit polyclonal anti-N-cadherin antibody (1:100 dilution; Abcam); goat polyclonal anti-CD24 antibody (1:20 dilution; Santa Cruz Biotechnology, Dallas, TX, United States); mouse monoclonal anti-CD44 antibody (1:50 dilution; Dako Co); mouse monoclonal anti-CD133 antibody (1:10 dilution; Miltenyi Biotec, Gladbach, Germany); and mouse monoclonal anti-EpCAM antibody (1:500 dilution; Cell Signaling).