Texred conjugated secondary antibodies

Cells were seeded on glass coverslips pre-coated with poly-L-lysine (Sigma-Aldrich, St. Louis, MO, USA) and allowed to attach for 48 h before treatment. For immunofluorescence staining, the cells were washed with PBS, fixed in 2% paraformaldehyde for 10 min at room temperature, washed with PBS again, and additionally fixed with ice-cold methanol for 10 min. Methanol was removed with PBS washing (5 times for 5 min). Alternatively, washed cells were fixed in 4% paraformaldehyde in PBS for 30 min at 4 °C and permeabilized with 0.5% Triton X-100 for 5 min. The cells were blocked for 30 min by using 10% normal goat serum and further incubated with primary antibodies for overnight at 4 °C. The next day the cells were washed 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. Finally, a DAPI stain (Sigma-Aldrich, St. Louis, MO, USA) was used for 1 min to outline the nuclei; the coverslips were washed with PBS twice and mounted on glass slides. The cells were visualized on an Olympus BX63 fluorescence microscope. Images were captured using a Spot advanced imaging system or a Nikon N-SIM confocal system.

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.