For immunofluorescence analysis, MCF-7 cells were fixed and permeabilized using ice cold 100% Acetone for detection of endogenous N-Ras using Anti-N-Ras Recombinant Rabbit Monoclonal Antibody (Product # 703435, 1:100 dilution) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of N-Ras protein (green), Panel b) is stained for nuclei (blue) using ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). Panel c) represents cytoskeletal F-actin staining using Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating nmembrane localization of N-Ras. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.