ab64943 staining GRK5 in Rat H9C2 cells by ICC/IF (Immunocytochemistry/immunofluorescence). H9C2 cells were infected with Ad and were treated with GRK5 and PFA fro 24 hours. Cells were fixed with paraformaldehyde, permeabilized with 1% Triton X-100 and blocked with 5% serum for 1 hour at room temperature. Samples were incubated with primary antibody (1/200 in 5% Goat serum) for 2 hours. An undiluted Alexa Fluor® 488-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody.See Abreview
Anti-GRK5 antibody (ab64943) at 1/500 dilution + HEK293 whole cell lysate at 30 µgSecondaryHRP-conjugated Goat anti-rabbit IgG polyclonal at 1/4000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.
All lanes : Anti-GRK5 antibody (ab64943) at 1/500 dilutionLane 1 : HUVEC extractLane 2 : HepG2 cell extractLane 3 : HeLa cell extractLane 4 : HeLa cell extract with immunising peptide
ICC/IF image of ab64943 stained HeLa 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 (ab64943, 1µg/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 ab64943 staining in human breast carcinoma 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 ab64943, 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.