ICC/IF image of ab63638 stained HeLa cells. The cells were 4% formaldehyde (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 (ab63638, 5µg/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.
Anti-PKC epsilon antibody (ab63638) at 1/500 dilution + Human Cell Lysate- Whole cell (HMEC) at 20 µgSecondaryDonkey anti rabbit Conjugation: HRP at 1/5000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.
Anti-PKC epsilon antibody (ab63638) at 1/500 dilution + Mouse Cell lysate - whole cell (NIH3T3) at 20 µgSecondaryDonkey anti rabbit HRP linked Conjugation: HRP at 1/5000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.
IHC image of PKC epsilon staining in human normal pancreas 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 ab63638, 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.