β catenin

Fixed samples were processed, paraffin-embedded, 4μm-sectioned and stained with a standard hematoxylin and eosin (H&E). For tumor progression in inoculated groups, the following parameters were taken into account:

Signs of macroscopic or microscopic pulmonary metastasis, liver weight, proliferative index (PI) (Ki-67, Master Diagnostica, Granada, Spain) and expression of β-catenin (Dako, Madrid, Spain).

Characterization of non-parenchymal cells and other factors related with inflammation or immunoregulation: Kupffer cells (KCs) (anti-rat CD68, Millipore, Madrid, Spain), COX-2 (Thermo, Madrid, Spain), arginase-1 (Santa Cruz Biotech.) and T-CD3 lymphocytes (Dako).

Vasculogenic factors: expression of VEGF (Abcam, Cambirge, UK) and HIF1-α (Santa Cruz Biotech., California, USA).

PI, KCs and T-CD3 lymphocyte quantities were estimated by averaging positive cell numbers in 10x high power fields (400x). All immunohistochemical procedures were performed by using an automated staining system (Dako), in accordance with the manufacturer protocols.

The following primary antibodies were used: mouse monoclonal antibodies to α-tubulin (clone DM1A, IgG1, Invitrogen); β-tubulin (clone TBN06, Invitrogen); h-β-I-tubulin (IgG2b, R&D Systems, Minneapolis, MN, USA); h-β-III-tubulin (IgG2a, R&D Systems); pan-actin (clone C4, Cell Signaling Technology Inc., Danvers, MA, USA); β-actin (IgG1, MCA5775GA, AbD Serotec, Kidlington, UK); γ-actin (IgG2b, (MCA5776GA, AbD Serotec); β-catenin (IgG1, DAKO, Glostrup, Denmark); E-cadherin (IgG2a, BD Transduction); MDR-1 (Santa Cruz Biotechnology, Dallas, TX, USA); N-cadherin (Cell Signaling Technology Inc., Danvers, MA, USA); claudin-1 (Cell Signaling Technology Inc.); and vimentin (V9, DAKO).
The following secondary antibodies were used: AlexaFluor488-, Red-X, AlexaFluor594-conjugated goat anti-mouse IgG or specific to IgG1, IgG2b, IgG2a, absorbed to other IgG isotypes, and goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA). DAPI (D9542, Sigma-Aldrich) was applied for DNA (nuclear) staining. HRP-conjugated secondary antibodies were used for Western blot analysis: anti-mouse IgG and anti-rabbit IgG (Santa Cruz Biotechnology).