Immunostaining and Imaging of Fly Brains

Dissection of fly brains was performed as previously described (Kondo et al., 2020 (link)) with minor modifications. Brains of female flies were dissected in PBS, pre-fixed in 1% paraformaldehyde (PFA) in PBS on the ice for up to 30 min, then fixed in 2% PFA in PBS for 1 h at room temperature. Fixed brains were washed in PBT (0.1% Triton X-100 in PBS) for 3 × 10 min. Immunostaining was performed by Kondo et al. (2020) (link). The following primary antibodies were used at the indicated dilution: rabbit anti-GFP (1:1,000; Invitrogen; A11122), mouse anti-TH (1:100; ImmunoStar Inc.; 22941), rabbit anti-CCHa2 (1:1,000) (Ida et al., 2012 (link)), and rat anti-N-cadherin (DN-EX #8; 1:100; Developmental Studies Hybridoma Bank). The following secondary antibodies were used at the indicated dilution: AlexaFluor-488 goat anti-rabbit (1:1,000; Invitrogen; A11034), Cy3 goat anti-rabbit (1:200; Jackson Labs), Cy3 goat anti-rat (1:200; Jackson Labs), and AlexaFluor-568 goat anti-mouse (1:1,000; Invitrogen; 11004). 86% Glycerol was used as a mounting medium, and either native or immunostained fluorescence was imaged.