Rabbit anti γ tubulin

Proliferating LCLs were centrifuged onto poly-l-lysine-coated coverslips, fixed, permeabilised, blocked and stained as described (Prodosmo et al., 2013 (link)). The following Abs were employed: mouse monoclonal anti-p53 (DO-7 1:100, DAKO, Santa Clara, CA, United States), rabbit anti γ-Tubulin (1:800, Sigma-Aldrich, St. Louis, MO, United States); 488-conjugated goat anti-rabbit and 594-conjugated goat anti-mouse polyclonal Abs (both 1:400, Alexa-Flour, Invitrogen). Immunofluorescence signals were examined through an Olympus BX53 microscope equipped with epifluorescence and photographs were taken (×100 objective) using a cooled camera device (ProgRes MF). p53-mitotic centrosome localization (p53-MCL) was assessed in >70 mitotic cells/sample as previously described (Prodosmo et al., 2013 (link)).

Cells were fixed in either 4% PFA for 20 min, in ice-cold methanol for 10 min (for γ-tubulin and centrin antibodies) or for 10 min in 1× PTEMF extraction buffer (20 mM PIPES, pH 6.8, 10 mM EGTA, 1 mM MgCl 2, 0.2% Triton X-100 and 4% PFA) prepared fresh from 4× stock solution (for all the mitotic checkpoint antibodies). Cells were permeabilized by 2×3 min wash in 0.1% Triton-X-PBS (PBS-TX), followed by blocking in PBS-TX containing 1% BSA for 30 min at room temperature, before being processed for immunofluorescence. Primary antibodies were diluted in 1% BSA in 1× PBS-TX in a 37°C incubator for 1 hr. Primary antibody dilutions were as follows: mouse anti-α-tubulin (DM1α, Sigma, 1∶1000), rabbit anti-γ-tubulin (Sigma, 1∶1000), rabbit anti-pericentrin (Bethyl, 1∶300), mouse anti-centrin-3 (Abnova, 1∶500), rabbit anti-TPX2 (Bethyl, 1∶100), mouse anti-Hec1 (GeneTex, 1∶100), human anti-centromere antibody (ACA, 1∶2000), mouse anti-BubR1 (Millipore, 1∶50), mouse anti-Mad1 (a kind gift from Dr. Andrea Musacchio, 1∶300), sheep anti-Mad2 (a kind gift from Dr. Steven Taylor's Laboratory, 1∶200), rabbit anti-Zwint-1 (Bethyl, 1∶100). Secondary antibodies conjugated to FITC, TRITC or Cy-5 (all from Jackson Laboratories) were chosen as appropriate and used as recommended by the supplier. DNA was counterstained with DAPI.

The primary antibodies were mouse anti-β-catenin (1:1000, BD Transduction Laboratories 610153), rabbit anti-β-catenin (1:1000, Sigma C2206), mouse anti-acetylated tubulin (1:1000, Sigma T6793), and rabbit anti-γ-tubulin (1:1000, Sigma T5192). Secondary antibodies were conjugated to Alexa Fluor dyes (goat polyclonal, 1:500, Invitrogen).

The brains were removed, post-fixed in 4 % paraformaldehyde in PB for 3 days, and cryoprotected in PB with 30 % sucrose for 3 days. Coronal sections of the brain (40 μm thick) were blocked in PB with 10 % BSA, 0.3 % Triton X-100 for 45 minutes. Sections were incubated overnight at 4 °C with primary mouse anti-α-tubulin or rabbit anti-γ-tubulin (1:250; Sigma-Aldrich) for staining of spindles and centrosomes respectively, or rabbit anti-GFAP (1:1000; Dako Italia, Milan, Italy), rabbit anti-NeuN (1:400; EMD Millipore, Billerica, MA, USA), or rabbit anti-Iba1 (1:200; Wako Chemicals, Richmond, VA, USA) antibodies, identifying astrocytes, neurons, and microglia respectively. Slices were rinsed and incubated in PB containing 0.3 % Triton X-100 with Alexa Fluor 647 donkey anti-mouse or Alexa Fluor 647 donkey anti-rabbit secondary antibodies (1:500; Life Technologies) for 2 hours at RT. Before mounting, slices were incubated with DAPI (1:4000; Sigma-Aldrich) for 10 minutes. Immunofluorescence was observed with a laser confocal microscope (SP5; Leica) and images were acquired. Image analysis was performed with Leica Application Suite X software.

All cell lines were grown in Dulbecco's modified Eagle's medium supplemented with 10% Fetal Calf Serum. Antibodies used were as follows: mouse anti-γ-H2AX S139 (Milipore), rabbit anti-Rad51, goat anti-ATR and rabbit anti-Chk1 (Santa Cruz), rabbit anti-P-RPA S4/8 (Bethyl), mouse anti-RPA34, rabbit anti-γ-tubulin, mouse anti-α-tubulin, mouse anti-β-actin and rabbit anti-Mre11 (Sigma), rabbit anti-Chk1 S317, S345, S296, rat anti-RPA34 and rabbit anti-Histone H3 (Cell Signalling). Antibodies against hSSB1 were raised in sheep as described previously (18 (link)).