Lane 1 : Anti-PI 3 Kinase p85 alpha antibody [M253] (ab86714) at 1/1000 dilution
Lane 2 : Anti-PI 3 Kinase p85 alpha antibody [M253] (ab86714) at 1/2000 dilution
Lane 3 : Anti-PI 3 Kinase p85 alpha antibody [M253] (ab86714) at 1/4000 dilution

All lanes : A431 cell lysate

Predicted band size: 85 kDa
Observed band size: 85 kDa

Immunocytochemical labeling of PI 3 Kinase p85 in aldehyde-fixed and NP-40-permeabilized A431 cells. The cells were labeled with ab86714, then the antibody was detected using appropriate secondary antibody conjugated to DyLight^® 594.

IHC image of PI 3 Kinase p85 staining in Human skeletal muscle 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 (ab64236) (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab86714, 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.

ICC/IF image of ab86714 stained SKNSH cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum (ab7481) / 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 (ab86714, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96879, DyLight^® 488 goat anti-mouse IgG (H+L) (ab96871) 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.