Plasma membrane-associated pAktser473 is lost in response to targeted inhibition of the phosphatidylinositol 3-kinase pathway. Tumor sections from vehicle control (two upper panels) or MK-2206-treated (lower left panel) or BEZ253-treated (lower right panel) mice were stained with secondary antibodies alone (negative control; upper left panel) or with anti-pAktser473 (red) and total Akt (green). DNA was visualized by 4',6-diamidino-2-phenylindole (DAPI) staining. Images were obtained using identical settings in the confocal microscope using a 20× objective. The sections were first imaged with a near-infrared (NIR) scanner and subsequently imaged by confocal microscopy to detect the fluorescent-labeled antibodies in the visible light area. Treatment with MK-2206 or BEZ235 causes a marked reduction of pAktser473 levels, and loss of membrane localization compared with vehicle-treated controls. Numbers above the images refer to the individual tumor bearing mouse. All scale bars = 20 µm.