ICC/IF image of ab93628 stained DU145 cells. The cells were 4% formaldehyde 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 ab93628 at 10µg/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.
IHC image of NFAT4 staining in Human normal kidney formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ 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 ab93628, 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.
All lanes : Anti-NFAT4 antibody (ab93628) at 1 µg/mlLane 1 : Thymus (Human) Tissue Lysate - adult normal tissue (ab30146)Lane 2 : Kidney (Human) Tissue Lysate - adult normal tissue (ab30203)Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate Lane 4 : Ramos (Human Burkitt's lymphoma cell line) Whole Cell Lysate Lane 5 : Raji (Human Burkitt's lymphoma cell line) Whole Cell 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.