Lanes 1-2 : Anti-gamma H2A.X (phospho S139) antibody (ab2893) at 1/500 dilution
Lanes 3-4 : Anti-gamma H2A.X (phospho S139) antibody (ab2893) at 1/500 dilution (+ Histone H2A.X (phospho Ser 139) peptide ab15645)
Lanes 5-6 : Anti-gamma H2A.X (phospho S139) antibody (ab2893) at 1/500 dilution (+ Histone H2A.X non-phospho peptide - ab15646)

Lanes 1 & 3 & 5 : Control HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate Histone preparation
Lanes 2 & 4 & 6 : Colcemid treated HeLa whole cell lysate Histone preparation

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution (Goat polyclonal to Rabbit IgG (HRP))

Predicted band size: 17 kDa

Blocking peptides at 1ug/lane.

 

Immunofluorescence photomicrographs of genotoxic-treated (1μM during 3 h) human induced pluripotent stem cells (hiPSCs) and hESCs-derived neuroprogenitors (NP) performed immediately after CPT treatment (1μM during 3 h). The figure shows representative images of cells stained with primary antibodies against ATM phospho-serine1981 (pATM), histoneγH2AX, p53, p53 phospho serine 15 (p53pSer15). Nuclei were counterstained with DAPI. The scale bars represent 100 μm.

Histone  gamma H2A.X was detected using ab2893.

From Figure 2a of Garcia et al PLoS One. 2016; 11(3): e0152607. Published online 2016 Mar 31. doi: 10.1371/journal.pone.0152607

Reproduced under the Creative Commons licence: https://creativecommons.org/licenses/by/4.0/ 

 

ab2893 staining gamma H2A.X in human mammary tissue by Immunohistochemistry (paraffin embedded sections).Paraffin-embedded blocks were sectioned and mounted on frost-free slides. The 3-10 µm sections were deparaffinized in xylene and rehydrated through a series of graded alcohols. Slides were washed with 1× PBS and endogenous peroxidases were blocked with 1.5% hydrogen peroxide in 1× PBS for 20 minutes at 25°C. After three 5 minutes washes in 1× PBS, slides were incubated in blocking solution (1× PBS with 0.1% Triton X-100, 3% bovine serum albumin) with 5% normal donkey serum for 10 minutes at 25°C. Control (no primary antibody) and experimental slides were incubated overnight at 4°C, respectively, in blocking solution alone or blocking solution with ab2893 at 1/1000 dilution. Biotin-conjugated secondary antibody 1/200 was added and slides were incubated at 25°C for 30 minutes and then washed three times with 1× PBS. The ABC Peroxidase Staining kit (1/100 dilution of each Reagent A and B in 1× PBS) was applied at 25°C for 30 min. After 3 washes with 1× PBS, staining was visualized with peroxidase-sensitive Sigmafast 3,3'-Diaminobenzidine tablets. All slides were counterstained with 0.1% methyl green for 3 min at 60°C, dehydrated in ethanol, cleared in xylene and mounted with Permount. Images were obtained at 40× using a Leica DMI4000B confocal microscope with the Retiga 2000R digital camera. Exposure times were kept constant for all samples.

ICC/IF image of ab2893 stained UV treated HeLa (Human epithelial cell line from cervix adenocarcinoma) cells. The cells were 100% methanol fixed (5 min) then permeabilised using 0.1% PBS-Triton and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to further permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab2893 at 1 µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor^® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor^® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43 µM for 1 hour at room temperature.

Asynchronous HeLa cells were paraformaldehyde fixed and immunofluorescently labeled with ab2893 that had been preincubated with either 1) non-phosphorylated or 2) phosphorylated H2AX peptide. Identical exposure times were employed. The Merge images present the DAPI and ab2893 channels as red and green, respectively.  Scale bars represent 5µm.

1) Non-phosphorylated peptides

2) Phosphorylated peptides

HeLa (Human epithelial cell line from cervix adenocarcinoma) cells were incubated at 37°C for 3h with vehicle control (0 µM) and different concentrations of camptothecin (ab120115). Increased expression of γH2A.X (phospho S139) in HeLa cells correlates with an increase in camptothecin concentration, as described in literature.

Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 20µg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with ab2893 at 1 µg/ml and ab10475 at 1 µg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.

ab2893 staining γH2A.X in MALME-3M cells treated with terfenadine (ab120270), by ICC/IF. Increase of γH2A.X nuclear expression correlates with increased concentration of terfenadine, as described in literature.
The cells were incubated at 37°C for 6 hours in media containing different concentrations of ab120270 (terfenadine) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab2893 (10 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab2893 staining γH2AX (phospho S139) in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with camptothecin (ab120115), by ICC/IF. Increased nuclear expression of γH2AX (phospho S139) correlates with increased concentration of camptothecin, as described in literature.
The cells were incubated at 37°C for 3h in media containing different concentrations of ab120115 (camptothecin) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab2893 (10 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab2893 staining γH2A.X in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with SN 38 (ab141108), by ICC/IF. Increase of γH2A.X nuclear expression correlates with increased concentration of SN 38, as described in literature.
The cells were incubated at 37°C for 6 hours in media containing different concentrations of ab141108 (SN 38) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab2893 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab2893 staining γH2A.X in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with CPT 11 (Irinotecan) (ab141107), by ICC/IF. Increase of γH2A.X nuclear expression correlates with increased concentration of CPT 11 (Irinotecan), as described in literature.
The cells were incubated at 37°C for 6 hours in media containing different concentrations of ab141107 (CPT 11 (Irinotecan)) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab2893 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab2893 staining γH2A.X in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with 10-Hydroxycamptothecin (ab141071), by ICC/IF. Increase of γH2A.X nuclear expression correlates with increased concentration of 10-Hydroxycamptothecin, as described in literature.
The cells were incubated at 37°C for 6 hours in media containing different concentrations of ab141071 (10-Hydroxycamptothecin) in DMSO, fixed with 100% methanol for 5 minutes at -20°C and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab2893 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab2893 staining γH2A.X in weri cells treated with TMPyP4 tosylate (ab120793), by ICC/IF. Increase of γH2A.X nuclear expression correlates with increased concentration of TMPyP4 tosylate, as described in literature.
The cells were incubated at 37°C for 24 hours in media containing different concentrations of ab120793 (TMPyP4 tosylate ) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab2893 (1 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

Asynchronous HeLa (Human epithelial cell line from cervix adenocarcinoma) cells were exposed to 2Gy and permitted to recover for 30min.  Cells were paraformaldehyde fixed (4%), immunofluorescently labeled with ab2893 and counterstained with DAPI.  The merge image presents the DAPI and ab2893 channels as red and green, respectively.  The scale bar represents 5µm.