G6539

3~6 days adult flies were used for dissection in 0.015% Triton X-100 in 1 × PBS. Dissected brains were fixed in 4% paraformaldehyde (PFA, Cat#AR-0211, Dingguo Biotech, China) at RT for 20 min on a shaker. They were then washed with PBS and blocked in blocking buffer (1 × Normal Goat Serum in wash buffer) for 30 min at RT. After blocking, samples were incubated in primary antibodies in blocking buffer overnight on a shaker at 4°C. They were washed and incubated in secondary antibodies in blocking buffer for 2 hours on a shaker at RT. All washes were performed with 0.3% Triton X-100 in 1 × PBS for 3 × 20 min at RT. Primary antibody: rabbit anti-GFP (1:500, A11122, Invitrogen), mouse monoclonal nc82 (1:500, Developmental Studies Hybridoma Bank), anti-RFP (1:500, 600-401-379S, Rockland) and mouse monoclonal anti-GFP (1:500, G6539, Sigma). Secondary antibody: 488-goat anti-rabbit (1:200, A11008, Invitrogen), 647-goat anti-mouse (1:200, A21235, Invitrogen) and 555-goat anti-rabbit (1:200, A21428, Invitrogen). Imaging was performed on an Olympus FV1000 confocal microscope with 2.5 or 3 μm optical sections at a resolution of 1024 × 1024 pixels for labellum or 1024 × 800 pixels for brain. All images were processed with ImageJ software. More detailed information of antibodies were listed in Table 1.

Fly brains were dissected in cold 1X phosphate buffered saline (PBS) and fixed in 2% paraformaldehyde made in 1X PBS at room temperature for 1 h on a nutator, washed four times for 20 min each in PAT (1× PBS, 0.5% PBS Triton, 1% BSA) at room temperature, blocked for 1 h at room temperature with blocking buffer (PAT + 3% Normal Goat Serum) and incubated with primary antibodies, diluted in blocking buffer, overnight on a nutator at 4 °C. The primary antibodies used were Mouse-GFP (SIGMA-ALDRICH, G6539. 1:500 dilution), Rabbit-TH (EMD-Millipore, AB152, 1:200 dilution), and Rat-DN-cadherin (Hybridoma Bank DSHB, DNEX#8, 1:50 dilution). This was followed by four washes for 20 min each in PAT, and incubation overnight on a nutator at 4 °C with secondary antibodies diluted in blocking buffer. The secondary antibodies were all from Molecular Probe and used at 1:500 dilution: Alexa Fluor 488 anti-Mouse (A11029), Alexa Fluor 568 anti-Rabbit (A11036) and Alexa Fluor 633 anti-Rat (A21094). Brains were then washed four times for 20 min each in PAT at room temperature and one time for 20 min in 1× PBS and mounted with VECTASHIELD mounting medium (Vector Laboratories, H-1000). Samples were imaged on a Zeiss 800 confocal laser scanning microscope.