Overlay histogram showing HeLa cells stained with ab26350 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab26350, 1μg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was a mix of mouse IgG1 [ICIGG1], (ab91353, 1μg/1x106 cells), IgG2a [ICIGG2A], (ab91361, 1μg/1x106 cells), IgG2b [PLPV219], (ab91366, 1μg/1x106 cells), IgG3 [MG3-35], (ab18394, 1μg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

ab26350 staining gamma H2A.X in pig spermatocytes by Immunocytochemistry/ Immunofluorescence. Cells were fixed with formaldehyde, permeabilized with Triton x100 and blocking with 0.15% BSA was performed for 30 minutes at 250C was performed. Samples were incubated with primary antibody (1/100: in PBS + 0.15% BSA+0.1% Tween 20) for 12 hours at 25°C. An Alexa Fluor®488-conjugated goat monoclonal to mouse IgG was used at dilution at 1/100 as secondary antibody.See Abreview

ab26350 (1µg/ml) staining gamma H2A in human spleen, using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear staining .Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH6.1 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

ab26350 (1/500) staining gamma H2A.X (phospho S139) in HeLa cells (green). Cells were fixed in paraformaldehyde, permeabilised with 0.5% Triton X-100/PBS and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please refer to Abreview.See Abreview

All lanes : Anti-gamma H2A.X (phospho S139) antibody [9F3] (ab26350)Lane 1 : Molecular weight markerLane 2 : Cell lysates prepared from human Jurkat cellsLane 3 : Cell lysates prepared from human Jurkat cells treated with staurosporineLane 4 : Cell lysates prepared from mouse NIH3T3 cellsLane 5 : Cell lysates prepared from CHO-K1 cellsLane 6 : Cell lysates prepared from Rat-2 cells