Rabbit anti human vimentin

The bulk Panc-1 cells, the upper chamber cells, and the lower chamber cells lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Merck Millipore, USA). Membranes were blocked with 5% (w/v) bovine serum albumin (BSA) in TBST for 1 h at room temperature and incubated overnight with primary antibodies at 4°C. They were subsequently incubated with horseradish peroxidase-conjugated second antibodies. The immunoreactive bands were detected by chemiluminescence (ECL Plus, Merck Millipore) and relevant blots were quantified by densitometry using LANE-1D software. For immune detection, the primary antibody preparations used were as follows: rabbit-anti-human-CD24, rabbit-anti-human-ESA, and rabbit-anti-human- Bmi-1 were obtained from Santa Cruz Biotechnology (SantaCruz, CA, USA). Rabbit-anti-human-Oct-4, rabbit-anti-human-E-ca, rabbit-anti-human-Vimentin, rabbit-anti-human-N-ca, rabbit-anti-human-SHH, and rabbit-anti-human-β-catenin were obtained from Cell Signaling Technology (Boston, USA). Anti-β-actin was obtained from Abcam Company (Cambridge, Britain). The secondary antibody preparations either anti-rabbit or anti-mouse were purchased from Boster Biotechnology Company (Wuhan, China).

Cells were plated onto glass coverslips, fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 15 min. Blocking solution was applied for 1 h at room temperature. Primary antibodies, mouse anti-human E-cadherin (Cell Signaling Technology), and rabbit anti-human vimentin (Cell Signaling Technology), were applied at 4 °C overnight. FITC-conjugated goat anti-rabbit and Cy3-conjugated goat anti-mouse secondary antibodies were loaded and incubated for 1 h at room temperature. Immunostaining signals and DAPI-stained nuclei were visualized at room temperature using a confocal microscope (FV10i; Olympus) equipped with a 10 × /0.30 NA objective lens (Olympus) and FluoView software (version 4.3; Olympus).