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 (pH6, 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 haematoxylin 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.
All lanes : Anti-Histone H3 (phospho S10) antibody [mAbcam 14955] - ChIP Grade (ab14955) at 1 µg/mlLane 1 : Control HeLa Histone Prep 0.5ugLane 2 : Colcemid treated HeLa Histone prep 0.5ugLane 3 : Control HeLa Histone Prep 0.5ug with Human Histone H3 (tri methyl K9, phospho S10) peptide (ab15644) at 1 µg/mlLane 4 : Colcemid treated HeLa Histone prep 0.5ug with Human Histone H3 (tri methyl K9, phospho S10) peptide (ab15644) at 1 µg/mlLane 5 : Control HeLa Histone Prep 0.5ug with Human Histone H3 (unmodified ) peptide (ab7228) at 1 µg/mlLane 6 : Colcemid treated HeLa Histone prep 0.5ug with Human Histone H3 (unmodified ) peptide (ab7228) at 1 µg/mlLane 7 : Control HeLa Histone Prep 0.5ug with Human Histone H3 (phospho S10) peptide (ab11477) at 1 µg/mlLane 8 : Colcemid treated HeLa Histone prep 0.5ug with Human Histone H3 (phospho S10) peptide (ab11477) at 1 µg/mlLane 9 : Control HeLa Histone Prep 0.5ug with Human Histone H3 (phospho S28) peptide (ab14793) at 1 µg/mlLane 10 : Colcemid tre
Indian muntjac and HeLa cells were fixed in paraformaldehyde and labelled with ab14955 (1/2000 dilution) for 30 minutes. The above image contains an interphase and a prophase Indian muntjac cells immunofluorescently labelled with anti-PhosS10 (green) and counterstained with DAPI (red).
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).
ab14955 (1/1000) stainning a population of Human HeLa 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.See Abreview
All lanes : Anti-Histone H3 (phospho S10) antibody [mAbcam 14955] - ChIP Grade (ab14955) at 0.5 µg/mlLane 1 : Control HeLa Histone prepLane 2 : Colecemid treated HeLa Histone prepLane 3 : Control HeLa Histone prep with Human Histone H3 (unmodified ) peptide (ab7228) at 1 µg/mlLane 4 : Colecemid treated HeLa Histone prep with Human Histone H3 (unmodified ) peptide (ab7228) at 1 µg/mlLane 5 : Control HeLa Histone prep with Human Histone H3 (phospho S28) peptide (ab14793) at 1 µg/mlLane 6 : Colecemid treated HeLa Histone prep with Human Histone H3 (phospho S28) peptide (ab14793) at 1 µg/mlLane 7 : Control HeLa Histone prep with Human Histone H3 (phospho S10) peptide (ab11477) at 1 µg/mlLane 8 : Colecemid treated HeLa Histone prep with Human Histone H3 (phospho S10) peptide (ab11477) at 1 µg/mlLysates/proteins at 5 µg per lane.SecondaryRabbit Anti-Mouse IgG H&L (HRP) (ab6728) at 1/5000 dilution
All lanes : Anti-Histone H3 (phospho S10) antibody [mAbcam 14955] - ChIP Grade (ab14955) at 1/1000 dilutionLane 1 : Wild type 0-4 hour old fruit fly embryo lysateLane 2 : 0-4 hour old fruit fly embryo lysate expressing wee RNAiSecondaryAnti-mouse IgG, peroxidase-linked at 1/10000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.