Immunocytochemistry

In order to visualize neuropil structures in the brain preparations with successfully labeled neurons, an antiserum marking synaptic regions (SYNORF 1) was used in immunostaining experiments. The anti SYNORF 1 (Developmental Studies Hybridoma Bank Univerity of Iowa) was raised against fusion proteins composed of glutathione-S-transferase and the Drosophila SYN1 protein (SYNORF 1; Klagges et al., 1996), and it is reported to detect synaptic neuropil in various insect species, heliothine moths included (Berg et al., 2002; Kvello et al., 2009). After analyzing the iontophoretically stained neuron by confocal microscopy, the brain was rehydrated through a decreased ethanol series (10 min each) and rinsed in PBS. To minimize non-specific staining, the brain was submerged in 5% normal goat serum (NGS; Sigma, St. Louis, MO, USA) in PBS containing 0.5% Triton X-100 (PBSX; 0.1 M, pH 7.4) for 3 h at room temperature before being incubated in the primary antibody SYNORF1 at 1:100 in PBSX at 4°C for 5 days. Then, the brains were rinsed in PBS for 6 × 20 min before being incubated in the secondary antibody, Cy2-conjugated anti-mouse (dilution 1:300 in PBSX; Invitrogen, Eugene, OR, USA), at 4°C for 3 days. The brains were finally rinsed for 6 × 20 min in PBS, dehydrated with an ascending ethanol series, and mounted in methyl salicylate.