For immunofluorescence analysis, Insulin (200 ng/mL, 10 minutes) treated MCF-7 cells were fixed and permeabilized for detection of endogenous IRSpS636/639 using Anti-IRSpS636/639 Recombinant Rabbit Multiclonal Antibody (ab277770, 2 μ/mL) and labeled with Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor^® 488 conjugate (1/2000). Panel a) shows representative cells that were stained for detection and localization of IRSpS636/639 protein (green), Panel b) is stained for nuclei (blue) with DAPI. Panel c) represents cytoskeletal F-actin staining using Alexa Fluor^® 555 Rhodamine Phalloidin (1/300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of IRSpS636/639. Panel e) shows untreated cells with no signal. Panel f) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.