Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse teratoma tissue labelling Histone H3 with ab70550 at 1/1000 (0.2µg/ml). Detection: DAB.
IHC image of ab70550 staining Histone H3 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 ab70550, 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.
ab70550 staining Histone H3 in HeLa cells. The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab70550 at 2.5μg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat anti-rabbit AlexaFluor®488 secondary (ab150077) at 2 μg/ml (shown in green). AlexaFluor®350 WGA was used at a 1/200 dilution and incubated for 1h with the cells, to label plasma membranes (shown in blue). Nuclear DNA was labelled in red with 1.25 μM DRAQ5™ (ab108410), which was added to the secondary antibody mixture. A secondary only negative control is displayed, which indicates that the Histone H3 staining observed is due to primary antibody specificity and not to unspecific binding of the secondary antibody to the cells.
All lanes : Anti-Histone H3 antibody (ab70550) at 0.04 µg/mlLane 1 : Histones isolated from 293T cells at 50 µgLane 2 : Histones isolated from 293T cells at 15 µgLane 3 : Histones isolated from 293T cells at 5 µg
Detection of Human Histone H3 by Western Blot of Immunprecipitate. ab70550 at 1µg/ml staining Histone H3 isolated from 293T cells, immunoprecipitated using ab70550 at 3µg/mg lysate (1 mg/IP; 20% of IP loaded/lane). Detection: Chemiluminescence with exposure time of 1 second.