γ h2ax mouse monoclonal antibody

Paraffin-embedded sections were successively rinsed in Ottix-Plus (MicromMicrotec, Brignais, France), OttixShapper (MicromMicrotec), and water. The slides were then incubated in unmasking buffer (100 mM TrisBase, 10 mM EDTA, 0.05% Tween 20, pH 9) for 30 min at 99 °C, before being permeabilized in PBS, 0.2% Triton X-100. After washing (PBS, 0.1% Tween 20, 0.05% TritonX-100), and blocking (PBS, 0.2% milk, 5% FBS, 0.1% Triton X-100) for 10 min, the slides were incubated with a 1:50 γ-H2AX mouse monoclonal antibody (Millipore, Burlington, VT, USA) overnight at 4 °C, and then with a 1:500 anti-mouse IgG-Alexa Fluor 488 at 37 °C for 2 h (Thermo Fischer Scientific, Waltham, MA, USA). After 3 washes in PBS, 0.1% Tween 20, and two in PBS alone, slides were incubated in 1 µg·mL⁻¹ DAPI (Merck) for 10 min and finally mounted with Fluoromount^® (Merck). Two slides and at least 400 nuclei were analysed for each condition. The results were expressed as the mean number of foci per nucleus.

Conjugated 53BP1 rabbit polyclonal antibody was obtained from Novus (NB100-309AF488); BRCA1 mouse monoclonal antibody (5C-6934) from Santa Cruz; BRCA1 rabbit polyclonal antibody (07-434) and γH2AX mouse monoclonal antibody (2535291) from Millipore; rabbit polyclonal antibody (39117) from Active Motif; RAD51 rabbit polyclonal antibody (20-001) from Bio Academia; CtIP mouse monoclonal antibody (61141) and RPA rabbit polyclonal antibody (AB76420) from Abcam; RAP80 rabbit polyclonal antibody (14466), RIF1 rabbit polyclonal antibody (A300-569A), mouse monoclonal antibody (200-301-H50), and BrdU mouse monoclonal antibody (B5002) from Rockland; EdU Click-iT (C10338) from Sigma-Aldrich; Alexa 488 goat anti-mouse antibody (A11OC1) from Molecular Probes; Cy5 goat anti-mouse antibody (195-175-166), anti-rabbit antibody (111-175-144), and Cy3 goat anti-mouse antibody (115-165-146) from Jackson.

HEK293 transfected cells were grown on glass coverslips, irradiated with 2 Gy of X-rays, and harvested after 0.5, 2, and 24 h. Cells were fixed in 2% paraformaldehyde, permeabilized on ice for 5 min with 0.2% Triton X-100, and blocked in PBS/1% BSA (v/w) for 0.5 h at room temperature. Slides were incubated over night with 1 µg/mL γ-H2AX mouse monoclonal antibody (Millipore, Billerica, MA), and detected with an anti-mouse FITC-conjugated secondary antibody (Immunological Sciences, Rome, Italy). Confocal analysis was performed using LCS microscope (Leica Microsystems, Heidelberg, Germany). Quantitative analysis of γ-H2AX foci was carried out by counting foci in at least 50 cells/experiment, in two repeated experiments.