Mouse monoclonal anti e cadherin

Cells were lysed in a lysis buffer (10 mM KCl, 20 mM HEPES, 5 mM EDTA, 1% NP-40, 0.25% deoxycholate, pH 7.4) with protease and phosphatase inhibitors (1 mM Na3VO4, 10 mM NaF, 1 mM phenylmethanesulfonyl fluoride, 2 μg/ml aprotinin). Protein concentrations were measured by the BCA protein assay (Pierce, IL, USA). Equal amounts of the protein were denatured in sample buffer and then electrophoresed by 5-10% SDS-PAGE, transferred to the polyvinylidene fluoride (PVDF) membrane (no. IPFL00010; Merck Millipore, Darmstadt, Germany) under 100 V for 2 h and incubated with the following primary antibodies overnight at 4°C: anti-β-actin antibody (Cell Signaling Technology); Mouse monoclonal anti-E-cadherin (#14472, Cell Signaling Technology), rabbit polyclonal anti-β-catenin (ab6302, Abcam); mouse monoclonal anti-cycle D1 antibody(MA1-12296, Thermo Fisher Scientific); polyclonal anti-c-myc antibody (#9402, Cell Signaling Technology). The primary antibody incubation was followed by incubation with fluorescent Dye-tagged secondary antibodies that were 20,000-fold diluted in Odyssey blocking buffer (TBS) and an Odyssey Infrared Imaging System (LI-COR).

For immunofluorescence, cells were washed with PBS and fixed in 4% paraformaldehyde at room temperature for 30 min. Lung tissues fixed in 4% paraformaldehyde for 48 h were embedded in paraffin and cut into 4-μm thick sections. Non-specific binding was blocked using 10% normal goat serum (Sigma). Cells or lung sections were incubated with the following primary antibodies after being diluted in PBS with 1% bovine serum albumin at 4°C overnight: mouse monoclonal anti E-cadherin (Cell Signaling Technology, Inc.) and rabbit monoclonal anti-Vimentin (Cell Signaling Technology, Inc.). Then, cells or tumor sections were washed twice with PBS and incubated with secondary antibodies at 37°C for 30 min as follows: FITC-conjugated goat-anti-rabbit IgG (Abcam) or TRITC-conjugated goat-anti-mouse IgG (Sigma). The slides were mounted in mounting medium with 4′, 6-diamidino-2-phenylindole (DAPI; Vector Laboratories) and viewed with a live cell station (Delta Vision, API).

To evaluate differences in tumor cell proliferation, 6-µm-thick sections were blocked with horse serum, endogenous peroxidase was blocked with hydrogen peroxide and the slides were incubated overnight with rabbit monoclonal antibody to Ki67 (Cell Signaling, 1:400 dilution) or mouse monoclonal anti-E-cadherin (Cell Signaling, 1:100 dilution). Tissue slides were rinsed with TBS-Tween and incubated for 30 min with secondary antibody (Vectastain Elite ABC kit; Vector Laboratories, USA) then developed with diaminobenzidine (BD Pharmigen) and counterstained with hematoxylin. The samples were observed under an optical microscope with a 100 × immersion objective. Same number of pictures was taken per piece of tissue, and Ki67 positive cells were quantified in each field (5 independent fields were quantified per sample, n = 3 per group).