Normal goat serum (ngs)

After the dissected brain tissues were fixed in 4% formaldehyde at 4 °C overnight, they were washed with 0.1 M PBS twice for 15 min each (pH 7.4). Thick sections of the brains (30 μm) were cut with a vibrating blade microtome (VT 1200 S, Leica, Wetzlar, Germany), washed in 0.1 M PBS for 15 min, and then incubated in 0.1 M PBS containing 5% normal goat serum (NGS, Boster, China) for 1 h at room temperature. The primary anti-Dop1 and anti-AC2 (custom made, see “Protein preparation and Western blot analysis” section of Methods for details) was diluted at 1:300 in 0.1 M PBS containing 2% NGS. Incubation with primary antibodies lasted for 48 h. The tissues were washed with 0.1 M PBS three times for 15 min each and subsequently incubated with the mixture of two secondary antibodies, Goat anti-rabbit antibody Alexa fluor 488 (1: 500, A11034, Life Technology) and Goat anti-mouse antibody Alexa fluor 546 (1: 500, A11030, Life Technology), for 1 h at room temperature. After washing three times, the tissues were mounted in anti-fade fluorescence mounting medium. The negative serum of Dop1 and AC2 from rabbit and mouse were used as the negative control. The nucleus of locust brain is labeled by Hoechst33342 (Life Technology) to indicate the brain structure. The fluorescence was detected using a Zeiss LSM 710 confocal microscope (Zeiss, Oberkochen, Germany).