Sc 6278

To identify primary cultures of fibroblasts from contaminating cancer cells, the absence of epithelial markers and the presence of fibroblast-related mesenchymal markers were checked. Cytokeratin 19 (CK19), an epithelial marker; vimentin (VIM) and alpha-smooth muscle actin (ASMA), mesenchymal markers, were selected. CCA cell lines were used as the positive controls for CK19 detection. Cells were cultured for 24 h in 96-well plates and fixed with 4% paraformaldehyde for 15 min and permeabilized with 1% Triton X-100 for 1 min. Non-specific binding was blocked by 1% BSA in 1X PBS for ASMA and 5% FBS in 1X PBS for VIM and CK19. The mouse anti-human CK19 antibody (1:100 dilution; SC-6278, Santa Cruz Biotechnologies Inc.), mouse anti-human VIM antibody (1:500 dilution; SC-6260, Santa Cruz Biotechnologies Inc.), and mouse anti-human ASMA antibody (1:200 dilution; Sigma-Aldrich) were used. The primary antibody was incubated for 3 h at room temperature. After washing, goat anti-mouse IgG-Cy3 (Jackson Immunoresearch Laboratories Inc.) was added for 1 h at room temperature followed by adding Hoechst stain (Invitrogen). The signals were detected under an inverted fluorescence microscope.