Goat anti rabbit antibodies conjugated to alexa fluor 488

Tissues were next incubated in primary antibody solutions for one week at 4 °C. Antibodies raised in rabbits against ELH were obtained from J. Blankenship (University of Texas), while rabbit antibodies against αCDCP, APGWamide and MIP were all obtained from J. van Minnen (Vrije Universiteit of Amsterdam). References describing the antigens used for raising each antibody are provided in Table 1. Finally, antibodies raised in rabbits against FMRFamide were obtained commercially from ImmunoStar (Hudson, WI USA, Cat #20091). The dilutions of each of the primary antibodies are also given in Table 1.
Following primary incubation, the ganglia were washed four times over 8-12 hours in fresh blocking solution. Tissue was then incubated for 5 days at 4 °C in darkness in either goat-antirabbit antibodies conjugated to AlexaFluor488 (Molecular Probes, Eugene, OR, USA; Cat # A11008) or goat-anti-rabbit conjugated to Cy3 (Jackson ImmunoResearch, West Grove, PA, USA; Cat #111-165-003), both diluted 1:200 in blocking solution. The ganglia were then washed for 6-8 hours in blocking solution before being transferred to glycerol mounting medium (25% 0.05M Tris buffer in 75% glycerol with the addition of 2% n-propyl-gallate to minimize photobleaching).