Phospho histone h2ax antibody

Deparaffinized tissue sections (5 μM) were incubated in sodium citrate buffer (10 mM Na-citrate, 0.05 % Tween 20, pH=6.0) at microwave for 20 min to retrieve antigens. Then tissue sections were rinsed with PBS and dehydrated in 95% ethanol. Denaturation was conducted with hybridization buffer (as in TIF assay) containing FITC-conjugated telomere sequence (TTAGGG)₃-specific PNA probe for 7 min at 80˚C on a heat block. Slides were washed sequentially with 70% formamide / 0.6 × SSC, 2 × SSC, PBS, 0.1% PBST and incubated with blocking buffer (4 % BSA in PBST) for 30 min. Sections were incubated with phospho-histone H₂AX antibody (Cell Signaling) in blocking buffer at RT for 1 h and washed with PBST and incubated with Alexaflour 568 conjugated goat anti-Rabbit antibody in blocking buffer at RT for 1 h. Sections were washed sequentially with 0.1% PBST and PBS. The slides were mounted with Vectashield mounting medium with DAPI. Images were captured with a fluorescence microscope using a 100X objective. TIFs were quantified using Image J.

Cells were fixed with 3.7% formaldehyde, permeabilized with 0.1% Triton-X100, blocked with 0.1% BSA and incubated for 1 hour at room temperature with phospho-histone H2A.X antibody (9718, Cell Signaling Technology) followed by Alexa488 or Alexa647-labelled secondary antibody and DAPI as counterstain. Imaging was performed with a Leica TCS SP8 laser scanning confocal microscope (Boulder Grove Il).

DNA damage was assessed using an immunofluorescence staining protocol to detect γH2AX, an early marker of DNA damage. For this, 15,000 cells were seeded in each well of an eight-well glass slide chamber 24 h prior to the study. 24 h later, the cells were incubated with either cell culture medium (0 mg/mL control) or treated, in triplicate, with NPs at a concentration of 0.25, 0.5, or 1 mg of Ag per mL for 4 h. The cells were fixed with 4% paraformaldehyde, blocked, and incubated at 4 °C overnight with Phospho-Histone H2AX antibody (Cell Signaling, Danvers, MA). As positive controls, the cells were irradiated with a dose of 6 Gy delivered by a Precision X-Rad 320IX Biological X-Ray Irradiator 30 min prior to fixation. After washing with PBS, the cells were incubated with an anti-rabbit AF 594 secondary antibody for 1 h. Following mounting with Vectashield antifade mounting media containing DAPI, the slides were imaged at 20× using a Zeiss AxioObserver Z1 equipped with DAPI and RFP filters. ImageJ was used to analyze the fluorescence intensity of the RFP and DAPI images. The mean intensity ratio was determined by calculating the ratio of RFP and DAPI fluorescence intensities. Data are presented as the mean ratio of fluorescence intensity from control ± standard deviation.