Brain immunohistochemistry

FoxP immunostainings were performed in wildtype animals or in the GFP-tagged FoxP animals. Third instar larvae or freshly eclosed adult males were collected. Brains were dissected and fixed in cold methanol for 2 minutes, washed with PBS-T (PBS (phosphate-buffered saline) containing 0.3% Triton X-100) for 1 hour and blocked with PBS-T 10% normal goat serum (NGS) for 2 hours. Brains were incubated with primary antibodies in PBS-T 10% NGS for 2 nighs at 4°C: guinea pig-anti-FoxP (1/300) (gift from D. Deitcher [38], rat-anti-Elav (1/100), mouse-anti-Repo (1/100), mouse anti-Dac (1/100), mouse-anti-Fasll (1/10), mouse-anti-nc82 (1/50) (Developmental Studies Hybridoma Bank, University of Iowa), rabbit-anti-GFP 1/600 (A11122, Invitrogen), rabbit-anti-TH 1/100 (Ab 152, Millipore). After primary antibody staining, brains were washed five times in PBS-T at room temperature (RT). Secondary antibodies were diluted 1/500 in PBS-T 3% NGS and incubated for 38h hours at 4°C: goat anti-rabbit Alexa Fluor 488 (A11008, Molecular Probes), goat anti-mouse Alexa Fluor 488 (A11029 Life technologies), goat anti-rat Alexa Fluor 488 (A11006, Molecular Probes), goat anti-guinea pig Alexa Fluor 568 (A11075, Invitrogen). Finally, brains were washed five times in PBS-T at RT and mounted with ProLong Gold Antifade Mountant (Thermo Fisher). Images were obtained with Zeiss AxioImager Z2 fluorescent microscope equipped with an ApoTome (Carl Zeiss B.V.).