Fig. 5. Double-immunolabeling shows that the anterior commissural organ (ACO) does not possess allatostatin-like peptide or serotonin co-transmitters. (A) Co-labeling of the commissural ganglion (CoG) with antibodies to substance P (-Sub P) and allatostatin (-AST). Both the substance P (A1) and allatostatin (A2) antibodies give rise to extensive labeling within the CoG. However, superimposition of micrographs of the two labels (A3) shows that the ACO (as defined by the substance P label) is not allatostatin-immunopositive. All micrographs in A are brightest pixel projections of 25 optical sections taken at 2.0 µm intervals. (B) Co-labeling of the CoG with antibodies to substance P and serotonin (-5HT). As in the previous pairing, both the substance P (B1) and serotonin (B2) antibodies give rise to extensive labeling within the CoG. Again, superimposition of micrographs of the two labels (B3) shows that the ACO (as defined by the substance P label) is not serotonin-immunopositive. All micrographs in B are brightest pixel projections of 30 optical sections taken at 2.0 µm intervals. A1, A2, B1 and B2 are shown at the same scale. Likewise, A3 and B3 are shown at the same scale. It should be noted that in A3 and B3, the apparent co-localization of the immunolabels (yellow coloration) is an artifact of the projection of multiple optical sections spanning the entire thickness of the ganglia shown rather than true co-localization of the immunoreactivities in a common structure. Scale bars, 100 µm.