Sc 9068

Sections (4 μm) were cut from paraffin embedded tissue and non-specific binding sites were saturated for 1 h at 37°C with PBS, 5% BSA. Sections were incubated at 4°C overnight with primary antibodies diluted in PBS supplemented with 1% BSA. The primary antibodies used were directed against VIM (Santa Cruz sc-7557), FN (Santa Cruz sc-9068), α-SMA (Sigma A5228), or HPSE (Santa Cruz sc-25826). Primary antibodies were revealed with an anti-goat-FITC for VIM, anti-rabbit-Cy3 for FN and HPSE, and anti-mouse-TR for α-SMA by incubation at room temperature for 45 min. Cell nuclei were visualized by Hoechst 33258. Images were obtained with a confocal LeicaSP5 microscope. Image processing was done with Image J (https://imagej.nih.gov/ij/).

Cells from cultures were lysed in RIPA buffer for HIF-1 alpha investigation and in 50 mM Tris-HCl, pH 5.0, 150 mM NaCl,0.5% Triton X-100 with Complete Protease Inhibitor Mixture (Roche Applied Science) for the other cytoplasmic protein analyses. Briefly, equal amounts of proteins were treated in reducing sample buffer and denatured for 10 min at 100°C. Protein samples were then resolved in 10% SDS-PAGE and electrotransferred to nitrocellulose membranes. Non-specific binding was blocked for 1 h at room temperature with non-fat milk (5%) in TBST buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20). Membranes were exposed to primary antibodies directed against GAPDH (Santa Cruz sc-25778), FN (Santa Cruz sc-9068), α-SMA (Sigma A5228), HPSE (InSight HP130) or HIF-1 alpha (BD 610959), overnight at 4°C and incubated with a secondary peroxidase-conjugated antibody for 1 h at room temperature. The signal was detected with SuperSignals West Pico Chemiluminescent substrate solution (Pierce) according to the manufacturer’s instructions.

Sections (4 μm) were cut from paraffin embedded tissue and non-specific binding sites were saturated for 1 h at 37°C with PBS, 5% BSA. Sections were incubated at 4°C overnight with primary antibodies diluted in PBS supplemented with 1% BSA. The primary antibodies used were directed against vimentin (Santa Cruz sc-7557), fibronectin (Santa Cruz sc-9068), α-SMA (Sigma A5228), or heparanase (Santa Cruz sc-25826). Primary antibodies were visualized with anti-mouse AlexaFluor-488, anti-goat-FITC, anti-rabbit-Cy3, and anti-mouse-TR secondary antibodies. Cell nuclei were visualized by Hoechst 33258. Images were obtained by a confocal LeicaSP5 microscope [35 (link)].