All lanes : Anti-Histone H3 (phospho S10) antibody (ab5176) at 1/5000 dilution

Lane 1 : Untreated HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa cell lysate treated with calyculin A
Lane 3 : HeLa cell lysate treated with calyculin A and alkaline phosphatase

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/20000 dilution

Predicted band size: 17 kDa


Exposure time: 1 second

Images of mouse cardiomyocyte in various stages of mitosis. Puromycin-resistant cells were stained with Hoechst 33342 (blue), and probed with antibodies specific to cTnT (Green) and Histone H3 (phospho S10) (Red) using ab5176 at 1/200 dilution in ICC/IF.

Mitotic activity was confirmed by staining with an antibody to phospho-H3 Histone, which is only phosphorylated at Ser10 during mitosis. Mitotic nuclei are pink (red+blue). Bar =10 µm.

IHC image of ab5176 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 ab5176, 5µg/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.

This image was kindly supplied by Prof Bryan Turner, University of Birmingham. Female Human Lymphoblastoid cells were incubated with ab5176.

All lanes : Anti-Histone H3 (phospho S10) antibody (ab5176) at 1 µg/ml

Lane 1 : untreated histones
Lane 2 : colcemid treated histones
Lane 3 : untreated histones with Human Histone H3 (phospho S10) peptide (ab11477) at 1 µg/ml
Lane 4 : colcemid treated histones with Human Histone H3 (phospho S10) peptide (ab11477) at 1 µg/ml
Lane 5 : untreated histones with Human Histone H3 (unmodified ) peptide (ab2903) at 1 µg/ml
Lane 6 : colcemid treated histones with Human Histone H3 (unmodified ) peptide (ab2903) at 1 µg/ml

Lysates/proteins at 0.5 µg per lane.

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

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 17 kDa


Exposure time: 10 seconds

ab5176 at a 1/100 dilution staining Drosophila melanogaster (wild type) polytene chromosomes by ICC/IF. The cells were formaldehyde fixed and blocked with 1% BSA prior to incubation with the antibody for 12 hours. Bound antibody was detected using a Cy3 conjugated goat anti-rabbit antibody.

See Abreview