Figure 2. Validation of Image Analytical Methods.

Three plane orthogonal views obtained from confocal Z-stacks of part of the rat PVHmpd. The arrangement of the X, Y, and Z planes are shown on panel a. The XY part of each panel shows the uppermost image of each Z-stack. The images show the degree of co-occurrence of structures immunostained with either a mouse monoclonal DBH antibody and a rabbit polyclonal DBH antibody DBH (a–d), or a mouse monoclonal DBH antibody and a rabbit polyclonal PNMT antibody (f–i). Panels a), b), f), and g) show structures stained with the individual antibodies. Panels c) and h) are the respective merged images where single labeled structures are in red or green and co-occurring structures in yellow. Panels d) and i) show any structure that contained labeling from both antibodies (white) extracted from panels c) and h), respectively. The horizontal and vertical white lines in panels a)–d) and f)–i) target a representative double-labeled structures in the X, Y, and Z planes. There is no significant difference between the percent co-occurrence of the structures labeled with the two DBH antibodies (e). However, because all PNMT always co-occurs with DBH but not all DBH occurs with PNMT there was a significant difference between the percent co-occurrence of the two antigens depending on which one was considered the reference antigen (j). Error bars in e) and j) indicate mean + S.E.M. Scale bar represents 20μm.