ab150291 stained HepG2 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 ab150291 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) 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.
All lanes : Anti-ATPB antibody (ab150291) at 1 µg/mlLane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell LysateLane 2 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell LysateLane 3 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell LysateLane 4 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell LysateLane 5 : RAW 264.7 (Mouse leukaemic monocyte macrophage cell line) Whole Cell LysateLane 6 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell LysateLane 7 : Liver (Mouse) Tissue LysateLysates/proteins at 10 µg per lane.SecondaryGoat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.
IHC image of ATPB staining in Human normal 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 ab150291, 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.