Immunofluorescence Microscopy of Mouse Anti-AKTpS473 antibody using STED nanoscopy to evaluate AKT activation and migration. Tissue: A431 cells. Antigen retrieval: Panel A: serum starved,unstimulated cells. Panel B: serum starved, EGF stimulated for 15 mins. A massive increase in AKT-pS473 activation, as measured by intensity signal, peaked at 15 minutes and was associated with depolymerized tubulin. Staining: Panel A shows STED data (AKT-pS473, red channel) collected simultaneously with confocal signal (a-tubulin, green channel). Upon stimulation of cells with EGF, a rapid activation of AKT is observed (Panel B) along with a coincident change in the tubulin organization (yellow signal), as well as an extensive cell shape-change (cell membrane folding) and accumulation of AKTpS473 at the cell periphery.