Brains were dissected and fixed in 4% (w/v) paraformaldehyde in PBS (1.86 mM NaH2PO4, 8.41 mM Na2HPO4, 175 mM NaCl) for 20–60 min at room temperature under vacuum. Samples were washed for 3×10 min with PBS containing 0.1% (v/v) Triton-X100 (PBT) and twice in PBS before mounting in Vectashield (Vector Labs). Images were collected on a Leica TCS SP5 confocal microscope and processed in Fiji.
APL expression of shits1, dTRPA1, and GADRNAi was scored by widefield imaging of mCherry (for functional imaging experiments) or GFP (for behavioral experiments) in unfixed brains mounted in PBS. APL expression of TeTx and TeTx-inactive was detected by immunostainings using rabbit anti-TeTx antibody (POL 016, Statens Serum Institut, 1:100) and goat anti-rabbit Alexa 546 conjugate (Jackson ImmunoResearch, 1:800). Primary and secondary antisera were applied for 2 days in PBT at 4 °C. The dsRed driven by the 3XP3 promoter in the GH146-Flp insertion62 (link) was not expressed in the mushroom body and therefore did not interfere with the detection of mCherry or TeTx in APL.
APL expression of shits1, dTRPA1, and GADRNAi was scored by widefield imaging of mCherry (for functional imaging experiments) or GFP (for behavioral experiments) in unfixed brains mounted in PBS. APL expression of TeTx and TeTx-inactive was detected by immunostainings using rabbit anti-TeTx antibody (POL 016, Statens Serum Institut, 1:100) and goat anti-rabbit Alexa 546 conjugate (Jackson ImmunoResearch, 1:800). Primary and secondary antisera were applied for 2 days in PBT at 4 °C. The dsRed driven by the 3XP3 promoter in the GH146-Flp insertion62 (link) was not expressed in the mushroom body and therefore did not interfere with the detection of mCherry or TeTx in APL.