Cyclin d1 primary antibody

CNE-2Z cells cultured on glass coverslips were fixed in paraformaldehyde (4%) for 30 min at room temperature, and then permeabilized with Triton X-100 (0.5%, v/v in PBS) at 4 °C for 5 min. After blocking with 5% normal goat serum for 60 min at room temperature, the cells were then incubated with the ClC-3 primary antibody (1:100, Abcam, USA) and cyclin D1 primary antibody (1:100, Cell Signalling Technology, USA) at 4 °C overnight. The next day, the coverslips were rinsed for 5 min with PBS three times and incubated with the secondary antibodies (1:100), Alexa Fluor® 405 (blue)-labeled goat anti-rabbit IgG and Cy3 (red)-conjugated goat anti-mouse IgG (Invitrogen, USA), in dark for 30 min at 37 °C. Between incubation steps, cells were rinsed with PBS. Cells were observed by the Nikon C1-si laser-scanning confocal microscope (Nikon instrumetns, Japan).

Proteins from glioma tissue samples and cell line (U251) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred on to polyvinylidene membranes. The blots were incubated with Cyclin D1 primary antibody (Cell Signaling, MA USA), c-Myc primary antibody (Cell Signaling), and β-catenin (Abcam, Cambridge, UK). Then, the blots were incubated with an appropriate second antibody that conjugated with the horseradish peroxidase for 2 h. Finally, the complexes were detected using chemiluminescence.