*4.6. Immunofluorescence Staining*

Cells were seeded on glass coverslips coated with poly-L-lysine (Sigma-Aldrich, St. Louis, Missouri, USA) and allowed to attach for 48 h before treatment. After washing with PBS, cells were fixed in 4% paraformaldehyde solution (in PBS) for 30 min at 4 ◦C and further permeabilized with 0.5% Triton X-100 for 5 min at 4 ◦C. Then the cells were incubated for 30 min in blocking solution containing 10% normal goat serum and 0.5% bovine serum albumin (in PBS). The cells were further incubated

for 30 min with the blocking solution containing 10% goat serum and 0.5% bovine serum albumin. After blocking procedure, the cells were incubated with primary antibodies for overnight at 4 ◦C. Next day the cells were washed twice with PBS, incubated with Alexa Fluor 488, or TexRed-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) for 30 min at room temperature in the dark. After brief DAPI (Sigma-Aldrich, St. Louis, MI, USA) staining, the coverslips were mounted on glass slides and visualized on fluorescence microscope "Olympus BX63" (Tokyo, Japan). Finally, the images were captured by using a Spot advanced imaging system.
