Synorf1

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.

For visualizing the neural architecture of the stained preparations, immunostaining with an antibody labeling presynaptic terminals was performed. The antibody SYNORF1 (Developmental Studies Hybridoma Bank, University of Iowa) was used as the primary antibody. The preparations were pre-incubated in 5% normal goat serum (Sigma, St. Louis, MO) in PBS containing 0.5% Triton X-100 (PBSX; 0.1 M, pH 7.4) for 3 h to minimize non-specific staining and then incubated in SYNORF1 at 1∶100 in PBSX at 4°C for 5 days. After rinsing in PBS for 6×20 min, the preparations were incubated in a Cy2-conjugated anti-mouse secondary antibody (Invitrogen, Eugene, OR; dilution 1∶300 in PBSX) at 4°C for 3 days. Finally, the preparations were rinsed 6×20 min in PBS, dehydrated with ascending ethanol series (50%, 70%, 90%, 96%, 2×100%, 10 min each), and mounted in methylsalicylate.