Mouse anti human vimentin monoclonal antibody clone v9

Cells were cultured on 8-well plastic chamber slides (Nunc^® Lab-Tek^® Chamber Slide™ system, Sigma Aldrich), fixed with 4% paraformaldehyde for 30 min and permeabilised using 0.1% Triton X-100 for 5 min. Slides were blocked with 10% horse serum/1% bovine serum albumin (1 h at room temperature). Mouse anti-human E-cadherin monoclonal antibody (clone 36, 1:100 dilution, BD Transduction Labs) or mouse anti-human vimentin monoclonal antibody (clone V9, 1:500 dilution, eBioscience, Thermo Fisher Scientific) were used with an Alexa-Fluor-568-conjugated anti-mouse secondary antibody (Molecular Probes). Nuclei were visualised using DAPI (Sigma Aldrich) and images generated using a LSM780 inverted confocal microscope (Carl Zeiss, North Ryde, NSW, Australia) with a 10× objective and associated ZEN software (Zeiss).

Orthotopic xenografts were resected from mouse mammary glands and fixed in 10% neutral buffered formalin for 4-8 h. Heat-induced antigen retrieval was conducted for 15 min on 4 µm sections submerged in citrate buffer (pH 6.0). Sections were blocked with 10% horse serum/1% bovine serum albumin (1 h at room temperature) and then incubated overnight (4°C) in primary antibody. Mouse anti-human E-cadherin monoclonal antibody (clone 36, 1:100 dilution, BD Transduction Labs), mouse anti-human vimentin monoclonal antibody (clone V9, 1:500 dilution, eBioscience, Thermo Fisher Scientific), or mouse anti-human pan-cytokeratin monoclonal antibody (clone PCK-26 ascites, 1:300 dilution, Sigma Aldrich) were used. PCK-26 recognises an epitope on the type II cytokeratins 1, 5, 6 and 8. Sections were incubated with a horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (Dako, Agilent Technologies, Mulgrave, Victoria, Australia) for 1 h at room temperature prior to timed incubation with the chromogen 3,3'-diaminobenzidine (Dako). Sections were counterstained with haematoxylin to visualise nuclei (blue) and slides were scanned using an Aperio Digital Pathology Slide Scanner (Leica Microsystems, Mount Waverley, Victoria, Australia). Images were generated using Aperio ImageScope software (Leica Microsystems).