ab109616 staining Hif-2-alpha in HeLa cells. The cells were incubated with 1mM Deferoxamine for 24 hours (Treated) or solvent-only for control purposes (Solvent only). Cells were fixed with 4% formaldehyde (10min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab109616 at 10µg/ml and ab195889 at 1µg/ml overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.
IHC image of HIF-2alpha staining in a section of formalin-fixed paraffin-embedded normal human colon*, performed on a Leica BOND. The section was pre-treated using heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 for 20mins. The section was then incubated with ab109616 at 1μg/ml, for 15 mins at room temperature. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.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.*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
All lanes : Anti-HIF-2-alpha antibody (ab109616) at 1 µg/ml (Milk blocking - 3%)Lane 1 : Placenta (Human) Tissue Lysate - adult normal tissue (ab29745)Lane 2 : A549 (Human lung adenocarcinoma epithelial cell line) Whole Cell LysateLane 3 : Heart (Human) Tissue Lysate - adult normal tissue (ab29431)Lane 4 : HeLa (Human epithelial carcinoma cell line) Whole Cell LysateLane 5 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell LysateLane 6 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate Lysates/proteins at 20 µg per lane.SecondaryGoat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.