ABSTRACT
The most common method used for measuring protein expression is quantitative immunoblot or western blot. However, in such approach, the cellular/tissue visualization of a specific protein is lost, since the protocol requires destructive cellular/tissue steps. This chapter describes a procedure to characterize protein expression regarding its level of expression and distribution in intra- and intercellular space. It provides the algorithm which includes a geometric compensation strategy to deal with cell morphological variability. The algorithm is composed of the following steps: nuclei selection; nuclei segmentation and centroid detection; fluorescence profile extraction from selected single cells or pairs of cells; image map building; intensity-preserved denoising of map image; geometric compensation of each 1D column profile; and computation of average and standard deviation profiles from compensated maps. The chapter illustrates the application of this methodology, used immunofluorescence (IF) real images from cancer cells stained for cell-cell adhesion proteins, such as E- and P-cadherins, and one of their molecular interactors, namely p120-catenin.
