Mouse monoclonal anti γ h2ax

MDA-MB-231 cells stably transfected with the miR-124 expression or control vector were seeded on coverslips, fixed for 15 min at room temperature in 3.7% paraformaldehyde in PBS, washed three times with cold PBS, and permeabilized for 10 min at room temperature in PBS containing 0.1% (v/v) Triton X-100. The cells were blocked in PBS containing 5% bovine serum albumin then labeled for 30 min at room temperature with mouse monoclonal anti-γ- H2AX (Ser139; 05–636; Millipore, Billerica, MA) and goat anti-mouse fluorescein isothiocyanate (Invitrogen). Nuclei were stained with 4', 6-diamidino-2-phenylindole. The coverslips were then mounted on microscope slides with an antifade reagent (VECTASHIELD Mounting Media; Vector Laboratories, Inc., Burlingame, CA) and images taken using an LSM700 confocal microscope (Oberkochen, Germany). At least 100 cells were assessed per coverslip, and cells were considered positive for γ-H2AX if they had >5 foci per nucleus.

Cells grown on coverslips were fixed with 2% paraformaldehyde, permeabilized with PBG-Triton and incubated with the primary antibody at 4°C overnight. The following day, cells were incubated with fluorescein-conjugated secondary antibody (Alexa Fluor 488 or 594; Invitrogen) for 45 min at room temperature. Primary antibodies used were as follows: rabbit polyclonal anti-Ki67 (ab15580; 4 μg/ml Abcam), mouse monoclonal anti-γH2A.X (no. 05–636; 0.25 μg/ml Millipore), and mouse monoclonal anti-p16 (SC-81156; 1:500 Santa Cruz).

Detection and scoring of immunofluorescence γ-H2AX and Rad51 in cell lines, was performed as previously described [15 (link), 27 (link)]. Xenografts were fixated in 3.6% paraformaldehyde (Aurion) and embedded in paraffin. Sections of 4 μm were prepared for detection of both cleaved-Caspase3 and γ-H2AX and heat-induced antigen retrieval was performed at pH 6. CASP3 sections continued with peroxidase blocking for 20 min and serum blocking using Ultra-V (Immunologic) for 5 min. Primary antibody Cleaved-Caspase3 (anti rabbit, Cell Signaling) was applied 1:200 overnight at 4°C. Thereafter, sections were incubated with Powervision Poly-HRP-GAM/R/R IgG (Immunologic) for 30 min and PowerDAB (Immunologic) for 1–2 min, counterstained with haematoxylin (Fluka) and mounted with pertex.
For γ-H2AX, sections were blocked after antigen retrieval in PBTB: PBS containing 0.1% Tween20 and 2% Bovine Serum Albumin (BSA) and incubated with primary antibody mouse monoclonal anti- γ-H2AX (Millipore) for 90 min (1:100 in PBTB) at room temperature. Secondary antibody goat anti-mouse Cy3 (Jackson Immunoresearch) was applied for 60 min, and DAPI (Sigma-Aldrich) was used as counterstain. After washing, sections were embedded in Vectashield and analysed by microscopy.