Immunostaining of Drosophila VNC

VNC were dissected in 1× PBS. Tissues were then fixed for 1 h in 4% formaldehyde in 1× PBS at room temperature. After fixation, brains and VNCs were washed three times (30 min per washing) in PBS with 0.3% Triton X-100 (PBTx) and incubated at 4°C overnight with primary antibodies. The following primary antibodies were used: mouse monoclonal anti-Ubx (FP3.38 (5 (link)) 1:500) from the Developmental Studies Hybridoma Bank), rabbit anti-TH (Novusbio), mouse anti-nc82, and chicken anti-GFP (Abcam Probes, 1:3,000). The secondary antibodies were anti-mouse Alexa Fluor 555 (Invitrogen Molecular Probes, 1:1,000), anti-rabbit Alexa Fluor 647 (Invitrogen Molecular Probes, 1:1,000), and anti-chicken Alexa Fluor 488 (Invitrogen Molecular Probes, 1:1,000). Images were acquired with a Leica SP8 confocal microscope, processed, and analyzed using FIJI ImageJ (NIH).