Immunohistochemical Localization of Vimentin, Fibronectin, α-SMA, and Heparanase

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)].