FIGURE 8.

Additional features of SynapseJ allow for identification of synaptic puncta with increased discrimination. (A) Specifying a minimum number of pixel overlap of 6 or more, removes some overlapping puncta (arrow), while other puncta remain (arrowhead), in this image of anti-bassoon (magenta) and anti-gephyrin (cyan) from BLA. Scale bar is 10 μm for the panels in panel (A). (B) In this image of anti-bassoon from SNc, applying the subtract background with a radius of 10 efficiently removed background staining while not modifying the brightly labeled puncta. Scale bar is 10 μm, and applies to panels in panels (B–D). (C) Original immunostaining for NR1 (cyan), tyrosine hydroxylase (blue) and YFP in SNc from the same area as the bassoon in panel (B). (D) The merge images for the original staining (left), identified overlapping anti-bassoon/anti-gephyrin puncta from the entire panel (center), and identified overlapping YFP⁺ PPN axonal anti-bassoon puncta with TH⁺ SNc neuronal anti-NR1 puncta (right) demonstrate the utility of SynapseJ in identifying neuronal sub-class limited expression of synaptic markers.