Cy2 conjugated anti mouse

Brains of A. dissimilis were prepared according to previous works (Zhao et al., 2016 (link)). Insects were decapitated and ALs were dissected in Ringer’s solution (Jiang et al., 2019 (link)) before transfer to 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS, pH 7.4) to be fixed at 4°C overnight. Brains were then rinsed in PBS (4 × 15 min) and preincubated with 5% normal goat serum (NGS; Sigma, St. Louis, MO, USA) in 0.1 M PBS containing 0.5% Triton X-100 (PBST; 0.1 M, pH 7.4) at 4°C overnight. SYNORF1 (Developmental Studies Hybridoma Bank, University of Iowa; Klagges et al., 1996 (link); Berg et al., 2002 (link)) primary antibody was used at a concentration of 1:100 (with 5% NGS in PBST) to stain the brains at 4°C for 5 days. The brains were later rinsed in PBS (6 × 20 min) and subsequently incubated with Cy2-conjugated anti-mouse (Invitrogen, Eugene, OR, USA; dilution 1:300 with 1% NGS in PBST) for 3 days at 4°C. Finally, the brains were washed in PBS (6 × 20 min) and then dehydrated 20 min for each concentration with ascending ethanol series (including 50%, 70%, 90%, 95%, and 100%) before being cleaned and mounted in methyl salicylate in a perforated aluminum slide with two glass coverslips.

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 (link)), and it is reported to detect synaptic neuropil in various insect species, heliothine moths included (Berg et al., 2002; Kvello et al., 2009 (link)). 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.

Brains were dissected in fresh Ringer’s solution (150 mM NaCl, 3 mM CaCl₂, 3 mM KCl, 25 mM Sucrose, and 10 mM N-tris (hydroxymethyl)-methyl-2-amino-ethanesulfonic acid, pH 6.9). In order to visualize the AL glomeruli, immunostaining with anti-synapsin antibody SYNORF1 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), was performed. The dissected brains were first fixed in a 4% paraformaldehyde solution in phosphate-buffered saline (PBS: 684 mM NaCl, 13 mM KCl, 50.7 mM Na₂HPO₄, 5 mM KH₂PO₄, pH 7.4) for 2 h at room temperature. After fixation, the brains were washed in PBS 4 × 15 min. To minimize non-specific staining, the brains were pre-incubated in 5% normal goat serum (Sigma, St. Louis, MO, USA) in PBS containing 0.5% Triton X-100 (PBSX; 0.1 M, pH 7.4) for 3 h 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 6 × 20 min, before being incubated in the secondary antibody, Cy2-conjugated anti-mouse (Invitrogen, Eugene, OR, USA; dilution 1:300 in PBSX), at 4°C for 3 days. The brains were finally rinsed 6 × 20 min in PBS, dehydrated with ascending ethanol series, and mounted in methylsalicylate.