Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 5µg of  ab12179 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.
    

IHC image of ab12179 staining Histone H3 (acetyl K9) in human colon 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 ab12179, 10µg/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.

ab12179 at a 1/1000 dilution staining Stylonychia lemnae (single cell organism, transcriptionally active macronucleus) by ICC/IF. The cells were paraformaldehyde fixed and incubated with the antibody for 12 hours. An Alexa Fluor ® 488 conjugated goat anti-mouse IgG antibody was used as the secondary.

In the image Histone H3 (acetyl K9) staining is red and is found in macronuclei only. Micronuclei remain unstained and are shown in blue (nucleic acid counterstain). Alpha tubulin is also stained (green).

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SK-N-SH cells fixed in 4% paraformaldehyde, permeabilized in 0.5% Triton X-100 and incubated for 1 hour with ab12179 (1/1000 dilution). ab12179 staining is localized to the nucleus (red). The cells were counterstained with DAPI (blue). 100x magnification.

All lanes : Anti-Histone H3 (acetyl K9) antibody [AH3-120] - ChIP Grade (ab12179) at 1 µg/ml

All lanes : Histone fraction isolated from HeLa cells

Secondary
All lanes : Goat Anti-Mouse IgG-Peroxidase