Anti phospho histone h2a x ser139 antibody

Tumors were fixed in 10% neutral buffered formalin (NBF) for 24–48 hrs, then processed and embedded in paraffin wax. 4 μm sections were cut onto electrostatically charged glass slides (Colorfrost®), de-waxed in xylene, and re-hydrated through graded alcohols. Heat induced antigen retrieval was performed using Dako Target Retrieval Solution (pH9.0) in a RHS-2 microwave (Milestone, Italy) at 110 °C for 2 minutes. Endogenous peroxide activity was blocked by immersion in 3% hydogen peroxide for 10 minutes and sections were subsequently incubated for 20 minutes with Background Blocker with Casein (A.Menarini Diagnostics Ltd, Berkshire) to reduce non-specific background staining. Immunohistochemistry was performed using a Labvision auto-stainer with X-Cell Plus polymer detection system (A.Menarini Diagnostics Ltd, Berkshire). Slides were incubated with rabbit polyclonal anti-phospho-histone H2AX (Ser139) Antibody (Cell signaling Technology), diluted 1:300 for 60 minutes at room temperature. Immunoreactivity was visualized using 3,3′ Diaminobenzidine (DAB) and slides subsequently counter-stained with haematoxylin.

Immunofluorescence was performed on adherent cells grown on coverslips, fixed in cold methanol for 7 minutes at -20°C and permeabilized with 0.15% Triton X-100 (T8787, Sigma-Aldrich) in PBS, blocked with 4% Bovine serum albumin (BSA) (A4503, Sigma-Aldrich)/PBS for 1 hour at room temperature and incubated with anti-phospho-histone H2A.X (Ser139) antibody (1:100, 9718, Cell Signaling Technology, Danvers, MA, U.S). Nuclei were counterstained by bisbenzimide Hoechst 33258 (0.5 µg/ml, B2883, Sigma-Aldrich, St. Louis, MA, U.S.). Cell fluorescence was then evaluated using a Nikon Eclipse 90i microscope (Nikon Instruments, Florence, Italy). For quantification purposes, γH2AX spots and micronuclei were quantified from at least 20 random fields (~500 cells), expressed as γH2AX mean fluorescence intensity (MFI) and percentage of cells with micronuclei respectively. Images acquisition and processing were conducted using the NIS-Elements A.R. 3,10 software.

D-17 cells were cultured on a 12-well plate and treated with CAP. After 24 h, cells were lysed with RIPA Cell Lysis Buffer (1X) with EDTA (GenDEPOT, Katy, TX, USA) supplemented with 100 μM of phenylmethylsulfonyl fluoride and sodium orthovanadate, respectively. Cell lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After transfer to a nitrocellulose membrane (GVS Filter Technology, Sanford, ME, USA), the membrane was blocked with 5% skim milk in Tris-buffered saline (TBS) with 0.05% Tween 20 (TBS-T) for 1 h, followed by incubation with an anti-phospho-Histone H2A.X (Ser139) antibody (1:1000) (2577S; Cell Signaling Technology, Danvers, MA, USA) overnight. Three washes with TBS-T were performed for 10 min each. The membrane was incubated with a secondary antibody conjugated with horseradish peroxidase (1:5000) for 1 h at room temperature, and it was washed again with TBS-T. Phospho-histone H2A.X was detected using an enhanced chemiluminescence (ECL) western blotting detection reagent (Thermo Fisher Scientific, Waltham, MA, USA). The western blot image was taken using Alliance Q9 mini (UVTEC CAMBRIDGE, Cambridge, England, UK), which was analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA). Anti-β-actin antibody (sc-47778; Santa Cruz Biotechnology, Dallas, TX, USA) was used for loading control.