ICC/IF image of ab151229 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 (ab151229, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 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 100% methanol fixed (5 min) Hek293, HepG2 and MCF7 cells at 1µg/ml, and in 4% formaldehyde fixed (10 min) HeLa, Hek293, HepG2 and MCF7 cells at 1µg/ml.
All lanes : Anti-ATP5A antibody (ab151229) at 1 µg/mlLane 1 : Brain (Human) Tissue Lysate - fetal normal tissue (ab29467)Lane 2 : Heart (Human) Whole Cell Lysate - fetal normal tissue Lane 3 : RAW 264.7 (Mouse leukaemic monocyte macrophage cell line) Whole Cell Lysate Lane 4 : HEK293 (Human embryonic kidney cell line) Whole Cell LysateLane 5 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate Lane 6 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell LysateLane 7 : HL60 (Human promyelocytic leukemia cell line) Whole Cell Lysate Lane 8 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell LysateLysates/proteins at 10 µg per lane.SecondaryGoat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.
IHC image of ATP5A staining in human heart muscle 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 ab151229, 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. 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.