Colocalization analyzed by merged pictures, scatter plots and correlation coefficient
Colocalization can be visualized in a merged picture of the two fluorescent signals. Overlapping pixels of the native GFP channel (green – figure 1A, 2A) and the channel of the antibody staining (red – figure 1B, 2B) appear in yellow (figure 1C, 2C). The merged pictures clearly show that the FluoTag®-X4 anti-GFP staining results in a uniform colocalization, whereas the conventional staining generates fluctuations in different compartments of the microglia. The nucleus shows a bright native GFP signal but only very faint antibody staining (figure 2A-C, star), which could be explained by worse penetration of the conventional antibodies into the nucleus or steric hindrance. In contrast, the processes of the microglia appear much brighter with the antibody staining compared to the GFP signal (figure 2A-C, arrow), most probably due to the higher amplification by secondary reagents. Scatter plots are another useful tool to visualize colocalization (ImageJ, Dunn et al., 2011). Here, the intensities of the colors are plotted against each other for each pixel (figure 1D, 2D). At high colocalization the points cluster around a straight line, as can be nicely seen in figure 1D.