All lanes : Anti-Histone H4 antibody (ab134212) at 1 µg/mlLane 1 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µgLane 2 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µgLane 3 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate at 10 µgLane 4 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µgLane 5 : HeLa Histone Preparation Nuclear Lysate at 2.5 µgLane 6 : Histone H4 Recombinant Protein at 0.1 µgLane 7 : Histone H3.1 Recombinant Protein (negative control) at 0.1 µgSecondaryGoat polyclonal Secondary Antibody to Chicken IgY - H&L (HRP) at 1/3000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.
All lanes : Anti-Histone H4 antibody (ab134212) at 1 µg/mlLane 1 : Histone H4 Recombinant ProteinLane 2 : Histone H2A Recombinant Protein Lysates/proteins at 10 µg per lane.SecondaryGoat polyclonal Secondary Antibody to Chicken IgY - H&L (HRP) at 1/3000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.
ICC/IF image of ab134212 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab134212 at 1µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- chicken IgY (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
IHC image of Histone H4 staining in human breast fibroadenoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol B. 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 ab134212, 5µg/ml, for 15 mins at room temperature. A goat anti-chicken biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. 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.