Distribution of endogenous (A) and heterologously expressed (B) VMAT2. A, Rat dorsal raphe neurons were prepared and cultured as described in Materials and Methods, fixed, and stained with primary antibodies directed against VMAT2 and MAP-2. Immunoreactive material was visualized with Alexa Fluor 488- and Alexa Fluor 568-conjugated secondary antibodies. Images were captured by confocal microscopy. A merged image is shown on the right to illustrate the fact that VMAT2 is found predominantly in the MAP-2-negative compartment. B, Rat dorsal raphe neurons were cotransfected with plasmids encoding GFP-tagged VMAT2 (green) and mCherry-tagged SERT (red) and stained for MAP-2 by using an Alexa Fluor 405-conjugated secondary antibody (blue). A 3D reconstruction was generated from z stacks (slice thickness, 0.5 μm) using NIH Image J (version 1.44p) to highlight the presence of both GFP-tagged VMAT2 and mCherry-tagged SERT in the MAP-2-negative compartment in close proximity to each other (white arrows). Scale bars, 20 μm.