Anti-Histone H3 (phospho S10) 抗体 [mAbcam 14955] (ab14955)

IHC image of ab14955 staining Histone H3 (phospho S10) in human kidney formalin fixed paraffin embedded tissue sections, performed on a Leica Bond.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab14955, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with hematoxylin and mounted with DPX. No primary antibody was used in the negative control (shown on the inset).

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

ab14955 staining Histone H3 (phospho S10) in HeLa cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab14955 at 1µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 100% methanol (5 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

Immunocytochemistry analysis of mouse 10T1/2 cells labeling Histone H3 (phospho S10) with ab14955 (1/2000 dilution) for 16 hours. Cells were fixed with paraformaldehyde and permeabilised with 0.5% Triton X100 in PBS. A Goat anti-Mouse Alexafluor488 at 1/200 was used as the secondary antibody. Cells were counterstained with DAPI. Dilution buffer: PBS.

The image demonstrates the mitosis associated staining pattern as the metaphase cell (centre), labels extremely well with the phosS10 antibody. This antibody also appears to give some interphase background staining as well.

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ab14955 (1/1000) stainning a population of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells positive for Histone H3 (phospho S10). Cells were trypsizined, pelleted and fixed in ice cold ethanol. Cellular debris was eliminated and the FL2-A/FL2-W was used to eliminate clumping cells. For further experimental details please refer to abreview.

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Flow cytometry dot plot showing HeLa cells stained with ab14955 (red line). The cells were fixed with 100% ethanol. The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab14955) (1x 10⁶ in 100μl at 1μg/ml (1/500)) for 30 min at 22°C. The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (ab150117) was used at 1/4000 for 30 min at 22°C. DNA staining was performed by pre-incubation with RNase A (250μg/ml) followed by DAPI.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

Indian muntjac and HeLa (Human epithelial cell line from cervix adenocarcinoma) cells were fixed in paraformaldehyde and labeled with ab14955 (1/2000 dilution) for 30 minutes. The image contains an interphase and a prophase Indian muntjac cells immunofluorescently labeled with anti-PhosS10 (green) and counterstained with DAPI (red).

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By ELISA, ab14955 detects:
the singly modified phospho S10 peptide and the dual modified phospho S10 and tri methyl K9 peptide (the orange and grey lines respectively),
less strongly detects the phospho S28 peptide (purple line),
does not detect the equivalent non-modified Histone H3 peptide for S10 or the singly modified tri methyl K9 peptide (the 2 lines at the bottom of the figure).

The antibody recognises phospho S28 by ELISA (although a phospho S28 peptide does not block the antibody in Western blotting).

Blocking: 10% BSA

wee shRNA embryos (lane 2) should display elevated phospho H3Ser10 levels relative to wild type

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