Anti γh2ax ser139 primary antibody

PBMCs were fixed with 1% formaldehyde fixation solution in phosphate-buffered saline (PBS) for 10 min at room temperature. Cells were rinsed three times with PBS and incubated with 0.1% Triton X-100 in PBS for 5 min and blocked with 2% BSA in PBS for 30 min. Cells were immunostained with anti-γH2AX (ser139) primary antibody (Merck) (1:3000, diluted in 0.2% BSA in PBS) at 4 °C overnight. After this incubation period, cells were rinsed three times with PBS and incubated with Alexa Fluor^® 488 anti-mouse secondary antibody (1:400, diluted in 0.2% BSA in PBS) for 1 h at room temperature. Cells were rinsed three times with PBS, and the coverslips were mounted in DAKO mounting medium on glass slides.
Confocal image stacks were captured with a Nikon C2+ microscope, using a 20× objective. Between 100 and 120 cells per patient were acquired. ImageJ analysis software was used to generate the zeta projections from 10 to 15 stacks (0.8 mm thickness each). Quantitative analysis of immunofluorescence data was carried out with a histogram analysis of the fluorescence intensity at each pixel across the nucleus. The data were normalized by the number of nuclei and are expressed in arbitrary fluorescence units (AU).

Cells were grown on Lab-Tek II 8-well glass slides (5 x 10⁴ cells/well) for 24 h and then exposed to ¹¹¹In-EGF-LP-Dox, ¹¹¹In-EGF-LP or EGF-LP-Dox for 24 h. Cells were then washed and fixed with 4% paraformaldehyde, permeabilised using 1% Triton X-100, blocked for 1 h using 2% BSA in PBS, and immunostained for γH2AX using an anti-γH2AX (Ser139) primary antibody (EMD Millipore, Hertfordshire, UK) and Alexa fluor 488 (AF488)-labelled secondary antibody. Cell nuclei were stained using DAPI and γH2AX foci were observed using a Zeiss LSM 780 confocal microscope. The number of γH2AX foci per cell was quantified using ImageJ software.