Anti phosphorylated histone 3

For immunocytochemical analysis of the cytoskeleton cells were plated at a density of 40000 cells/cm² on glass coverslip (VWR) in a 4 well plate (Nunc, Nunclon). For cilia staining, cells were plated at 50000 cells/cm² and after 22 h medium was changed to serum free medium. Cells were fixed using 4% paraformaldehyde on ice and subjected to primary antibody incubation overnight at 4°C. The primary antibodies were: anti-Arl13b (Proteintech, 1:1000), anti-acetylated α tubulin (Sigma, 1:10000), anti-α tubulin (Sigma, 1:10000), anti-pericentrin (Abcam, 1:1000) and anti-phosphorylated histone 3 (Cell Signalling, 1:1000). Secondary antibody and Hoechst (1:2000, Molecular Probe) incubations were done in 2% donkey serum in PBS at room temperature for 1.5 h. The secondary antibodies were: Alexa Fluor 488 (1:500, Molecular Probe), Alexa Fluor 594 (1:1000, Molecular Probe).

Cells were fixed in 4% formaldehyde for 10 min with shaking at room temperature, permeabilized for 5 min with 0.5% Triton X-100 in PBS, and blocked for 1 h at room temperature with 5% BSA in PBS containing 0.1% Triton X-100. The cells were labeled overnight at 4 °C with the primary antibodies anti-Ki67 (1:200, 275R, Cell Marque) and anti-phosphorylated histone 3 (1:200, 9701, Cell Signaling). After three washes with PBS, samples were labeled for 1 h at room temperature with fluorescent secondary antibodies (Abcam) followed by 10 min of DAPI staining for nuclear visualization. After three washes in PBS, cells were imaged with a Nikon Eclipse Ti2 microscope.