All lanes : Anti-Sprouty 2 antibody (ab85670) at 1 µg/mlLane 1 : Brain (Human) Tissue Lysate - adult normal tissue (ab29466)Lane 2 : Lung (Human) Tissue LysateLane 3 : Heart (Human) Tissue Lysate - adult normal tissue (ab29431)Lane 4 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate Lane 5 : Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate Lane 6 : SW480 (Human colon adenocarcinoma cell line) Whole Cell Lysate Lysates/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.
ICC/IF image of ab85670 stained PC12 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 (ab85670, 10µg/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 100% methanol fixed (5 min) PC12 cells at 10µg/ml.
IHC image of Sprouty 2 staining in human skin melanoma 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 ab85670, 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.