embedded in mounting medium and collected on a Zeiss LSM 510 confocal microscope (Zeiss, Oberkochen, Germany). To examine the specificity of immunostaining with the Ranti- AmOA1 serum, sections were incubated with the secondary antibody in the absence of primary antiserum (not shown), or they were immunoassayed with Ranti-AmOA1 serum (Fig. 1A) or preimmune serum (Fig. 1B). Furthermore, sections were incubated with antibody that had been preadsorbed with glutaraldehyde treated KLH (KLH-G) alone (Fig. 1C) or KLH-G conjugated to the AmOA1 peptide used to make antibodies in rabbit (Fig. 1D). To prepare the peptide conjugates for preadsorbtion, 100 mM of peptide was conjugated to KLH via gluteraldehyde [45]. For double immunofluorescence staining with Ranti-AmOA1 and with anti-GABA, sections labeled with Ranti-AmOA1 were detached from slides and washed in 0.05 M Tris-HCl, pH 7.5. They were then postfixed in 4% paraformaldehyde in PBS with 1% SMB for 20 minutes in order to deactivate the antibodies of the first sequence of staining. Then sections were processed for GABA immunostaining using the same protocol as described above. To compare octopamine (or GABA) immunostaining with anti-AmOA1 staining, we stained alternating sections from the same brain. For control of double immunostaining (Figure S1A) we used Tris-HCl buffer with 5% swine serum instead of GABA antiserum (Fig. S1B) or AmOA1 antiserum (Fig. S1C) on two consecutive sections. In these controls we did not observe any interaction between the reagents of the first and the second sequences of staining. AmOA1 in the Honey Bee Brain To test the specificity of GABA immunostaining, working dilutions of GABA antibodies were preincubated overnight with conjugate containing 100 mM hapten conjugated GABA-G-BSA. After preadsorption of the primary antiserum with GABA-G-BSA, the staining was abolished. Immunohistochemistry with primary antiserum from goat (Ganti-AmOA1) After reduction of double bonds with NaBH4, sections were washed (6610 min) in PBS buffer with 0.5% of Triton X100 (PBS- TX), then preincubated with 5% Normal Donkey Serum (Jackson ImmunoResearch) for one hour. The brain sections were then incubated overnight with affinity purified anti-AmOA1antibodies from goat (Ganti-AmOA1) diluted 1:100 in PBS-TX. After washing (6610 min) in PBS-TX, sections were incubated overnight in F(ab’) 2 fragments of donkey anti-goat IgG antibodies conjugated to either Cy2 or Cy3 (Jackson Laboratories, West Grove). After a final wash in PBS buffer, sections were embedded in mounting medium and data were collected on a Zeiss LSM 510 confocal microscope (Zeiss, Oberkochen, Germany). To examine the specificity of the immunostaining with the Ganti-AmOA1 antibodies, sections were incubated with the secondary antibody in the absence of primary antiserum (not shown) or they were immunoassayed with Ganti-AmOA1 (Fig. 1E) or preimmune serum (Fig. 1F). Furthermore, sections were incubated with antibody that had been preadsorbed with KLH Figure 1. Anti-AmOA1 antibodies are specific for the AmOA1 receptor. Pre- and post-adsorption controls of immunolabeling with anti- AmOA1 antibodies from rabbit (A–D) and from goat (E–G). Consecutive sections of the mushroom body calyx stained with Ranti-AmOA1 serum (A) and pre-immune serum taken from the rabbit before immunization and used at the same dilution as the Ranti-AmOA1 serum (B). The Ranti-AmOA1 serum revealed staining in Kenyon cell bodies (K) with different intensity and in processes of the calyx lip and collar (col). C: Section of the antennal lobe stained with Ranti-AmOA1 that was preadsorbed with KLH treated with glutaraldehyde (KLH-G) alone. D: A section from the same brain stained with Ranti-AmOA1 that was preadsorbed with the AmOA1 peptide conjugated to KLH (KLH-G-OA1pep). E: Sections of the antennal lobe stained with affinity purified anti-AmOA1 antibodies from goat (Ganti-AmOA1). F: pre-immune serum from the goat before immunization used at the same dilution as the Ganti-AmOA1 serum. G: Specific staining from cell bodies and their processes disappeared after pretreatment with the KLH conjugated to the AmOA1 peptide. The confocal images in D, F and G were adjusted to a higher level of intensity then stained sections in C, E to show the image of the antennal lobe. Scale bars: 50 mm. doi:10.1371/journal.pone.0014536.g001 PLoS ONE | www.plosone.org 3 January 2011 | Volume 6 | Issue 1 | e14536