Goat anti mouse alexa488

Adult flies were fixed with 4% paraformaldehyde in PBS/0.15% Triton X-100 for 2.5 h and brains were afterwards dissected. Larval brains were dissected first and then fixed in 4% paraformaldehyde in PBS for 30 min. Brains were washed twice with PBT (0.3% Triton in PBS) before blocking in 5% normal goat serum (NGS) (5% NGS in PBT) at room temperature for 1 h, followed by incubation with primary antibodies at 4°C overnight. Primary antibodies were diluted in 5% NGS: mouse anti-TH clone LNC1 (1:200, Merck, MAB318), guinea pig anti-Lamin-DmO (1:300, kind gift from Georg Krohne, University of Würzburg, Würzburg, Germany), mouse anti-Fas2 (1:10, Developmental Studies Hybridoma Bank) or rabbit anti-GFP (1:1000, MoBiTec, A6455). Following three washing steps in PBT, brains were incubated for 4 h at room temperature with secondary antibodies: donkey anti-guinea-pig-DL650 (1:100, Thermo Fisher Scientific, SA5-10097) or donkey anti-guinea-pig-Cy3 (1:100, Dianova, 706-166-148); goat anti-rabbit-Alexa488 (1:100, Molecular Probes, A-11034); goat anti-mouse-Alexa488 (1:150, Dianova, 115-545-166), donkey anti-mouse-Cy3 (1:100, Dianova, 715-165-151) or donkey anti-mouse-Cy5 (1:100, Dianova, 715-175-151). After washing in PBT, brains were embedded in VectaShield (Vector Laboratories) and confocal images were recorded with a Leica TCS SPE microscope.