All lanes : Anti-FOXO3A antibody (ab23683) at 1 µg/mlLane 1 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate (ab7899)Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate (ab7899) with Human FOXO3A peptide (ab24031) at 1 µg/mlLysates/proteins at 20 µg per lane.
All lanes : Anti-FOXO3A antibody (ab23683) at 1 µg/mlLane 1 : Heart (Mouse) Tissue Lysate - normal tissueLane 2 : Skeletal Muscle (Mouse) Tissue Lysate (ab29711)Lane 3 : Heart (Rat) Tissue Lysate - normal tissueLysates/proteins at 10 µg per lane.SecondaryIRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution
ICC/IF image of ab23683 stained MCF7 cells. The cells were 4% PFA fixed (10 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 (ab23683, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (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. This antibody also gave a positive result in 4% HepG2 fixed (10 min) HepG2 cells at 5µg/ml.
IHC image of FOXO3A staining in Human normal lung formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. 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 ab23683, 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.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.