Fibronectin coated coverslips

For immunofluorescence staining, cells were grown on fibronectin-coated coverslips (Becton Dickinson, Bedford, MA). Cells were fixed with methanol, permeabilized with 1% NP-40, and blocked with 10% BSA, followed by incubating with primary antibody. After washing, cells were incubated with FITC-labeled secondary antibody. Finally, coverslips were washed and mounted using Vectashield (Vector Laboratories, Burlington, CA). Stained slides were examined under a fluorescence microscope. Cells without primary antibody, or with Isotype-specific control IgG, were used as negative controls. Immunohistochemistry of tissues was performed as described elsewhere^{35 (link)}.

For immunofluorescence staining, cells were grown on fibronectin-coated coverslips (Becton Dickinson, Bedford, MA). Cells were fixed with methanol, permeabilized with 1% NP-40, and blocked with 10% BSA, followed by incubating with primary antibody. After washing, cells were incubated with FITC-labeled secondary antibody. Finally, coverslips were washed and mounted using Vectashield (Vector Laboratories, Burlington, CA). Stained slides were examined under a fluorescence microscope. Cells without primary antibody, or with Isotype-specific control IgG, were used as negative controls. Human pancreatic normal and cancer tissue arrays were purchased from US Biomax, Inc. (Rockville, MD). Immunohistochemistry of human normal and tumor tissues was performed as described elsewhere [38 (link)].

CSCs were grown on fibronectin-coated coverslips (Beckton Dickinson, Bedford, MA), and exposed with rotenone (0 and 40 μM), washed in PBS, and fixed for 15 min in 2% paraformaldehyde. Cells were permeabilized in 0.1% Triton X-100, washed and blocked in 10% normal goat serum. After blocking, cells were incubated with alexa fluor tagged antibody against CD9, CD63, CD81 and Alix (1:100) overnight at 4°C. Cells were washed with PBS and incubated with 4, 6-diamido-2-phenylindole hydrochloride (DAPI) (1 mg/ml) for 1 h at room temperature. Finally, coverslips were washed and mounted using vectashield (Vector Laboratories, Burlington, CA). Isotype-specific negative controls were included with each staining. Stained cells were mounted and visualized under Leica 6000B microscope with 100X objectives. The number of cells expressing punctate and the number of punctate per cell were counted under fluorescence microscope.