ICC/IF image of ab68826 stained HeLa cells. The cells were 100% methanol fixed (5 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 ab50599 5µg/ml overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti- rat 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.

Overlay histogram showing HeLa cells stained with ab50599 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab50599, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rat IgG (H+L) (ab98386) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rat IgG2a, kappa monoclonal [aRTK2758] (ab18450, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde/permeabilized in 0.1% PBS-Tween used under the same conditions.

All lanes : Anti-alpha Actinin antibody [MAC 276] (ab50599) at 1 µg/mlLane 1 : Skeletal Muscle (Human) Tissue Lysate - adult normal tissue (ab29330)Lane 2 : Heart (Rat) Tissue Lysate Lane 3 : Skeletal Muscle (Rat) Tissue Lysate Lysates/proteins at 10 µg per lane.SecondaryPeroxidase Conjugated AffiniPure Rabbit Anti-Rat IgG (H+L) at 1/10000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.