Ab26350 staining H2A.X  in Human placenta tissue sections by Immunohistochemistry (IHC-P - formaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 5% BSA for 30 minutes at 22°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/100 in TBS) for 16 hours at 4°C. A diluted Biotin conjugated Goat anti-mouse polyclonal (1/200) was used as the secondary antibody.

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All lanes : Anti-gamma H2A.X (phospho S139) antibody [9F3] (ab26350)

Lane 1 : Molecular weight marker
Lane 2 : Cell lysates prepared from Jurkat (Human T cell leukemia cell line from peripheral blood) cells
Lane 3 : Cell lysates prepared from Jurkat cells treated with staurosporine
Lane 4 : Cell lysates prepared from NIH/3T3 (Mouse embryonic fibroblast cell line) cells
Lane 5 : Cell lysates prepared from CHO-K1 (Chinese hamster ovary cell line) cells
Lane 6 : Cell lysates prepared from Rat2 (Rat fibroblast cell line) cells

This image was generated using the ascites version of the product.

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.

This image was generated using the ascites version of the product.

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.

This image was generated using the ascites version of the product.