All lanes : Anti-EDG8 antibody (ab92994) at 1 µg/mlLane 1 : Brain: corpus callosum (Human) Whole Cell Lysate - Alzheimer (ab30027)Lane 2 : Brain (Human) Tissue Lysate - adult normal tissue (ab29466)Lane 3 : Spleen (Human) Tissue Lysate - adult normal tissue (ab29699)Lane 4 : Lung (Human) Tissue Lysate Lane 5 : U937 (Human leukemic monocyte lymphoma cell line) Whole Cell Lysate Lane 6 : MOLT4 (Human acute lymphoblastic leukemia cell line) Whole Cell Lysate Lysates/proteins at 10 µg per lane.SecondaryGoat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP), pre-adsorbed at 1/5000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.
ICC/IF image of ab92994 stained PC12 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 ab92994 at 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.
IHC image of EDG8 staining in human hippocampus 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 sb92994, 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.