Primary rabbit polyclonal antibody to beta tubulin

Cells were grown on Nunc Labteck chamber slides for 24 h and then treated with 1 µM RDS 60 or DMSO for 24 h. Cells were washed with PBS with Ca/Mg (washing buffer) and fixed with 4% buffered paraformaldehyde (Sigma Aldrich) for 20 min at 4 • C, then permeabilized with PBS, 5% FCS, 0.5% TritonX100 for 30 min at RT and incubated for 1 h to RT with the primary rabbit polyclonal antibody to beta-tubulin (1:200 diluted; Immunological Sciences, Rome, Italy). Alternatively, cells were permeabilized with 0.1% TritonX100 for 10 min to RT, incubated with 3% BSA for 1 h at RT and incubated with the primary rabbit polyclonal antibody to cyclin B1 in 0.1% BSA (1:200 diluted; Elabscience, Houston, TX, USA) overnight at 4 • C. Cells were washed twice with washing buffer and incubated with the secondary anti-rabbit antibody FITC conjugated (1:400 diluted; Molecular Probes, Eugene, OR, USA) for 1 h at RT. Cells were washed twice with washing buffer and DNA was stained with Hoechst for 15 min at RT. The slide was mounted with ProLong-Antifade (Life Technologies, Carlsbad, CA, USA) and analyzed by a fluorescence microscope (Olympus BX52, Olympus America, Inc., Melville, NY, USA). Image acquisition and processing were conducted by IAS 2000 software.