Seven-color segmentation of a composite confocal image (3D projection) to analyze mRNA/protein and protein/protein co-localization in a rat brain slice. Scale bar: 10 μm. (a) Composite image (merged from three channels) taken from Fig. 3a. BDNF mRNA, Htt and Ago2 are shown in red, green and blue, respectively. (b) An artificial presentation of (a) to demonstrate distribution of different color pixels in (a). (c) Image in (a) was segmented using self-programmed software integrated in ImageJ to generate seven pseudo channels, each of which represents single or co-localized target(s). The secondary colors yellow, cyan and magenta indicate overlap of green (Htt) and red (mRNA), green (Htt) and blue (Ago2), red (mRNA) and blue (Ago2), respectively. The seventh pseudo channel is shown in (d). N: nucleus, S: soma. (d) White signal is the color addition product of three primary colors green, red and blue, representing co-localization of Htt, BDNF mRNA and Ago2. Due to the limitations in the co-localization analysis function of the LSM software for confocal microscope, we can calculate percent localization for double but not triple co-localization.