Mouse gfp

Brains and VNC of 5–7-days-old flies were dissected in dissection buffer (PBST: 0.015% triton X-100 in 1x PBS) and fixed in 4% PFA at room temperature on a shaker for 20 min, then washed for 4 × 20 min in wash buffer (0.3% triton in 1x PBS). After this, the tissues were blocked in block buffer (1x heat inactivated normal goat serum with 0.3% triton in 1x PBS) for 30 min at room temperature. The samples were then incubated with primary antibody at 4 °C overnight. The primary antibodies used were: Rabbit-GFP (Invitrogen, A11122, 1:500 dilution), Rabbit-DsRed (Rockland 39707. 1:500 dilution), Mouse-GFP (Sigma-Aldrich, G6539. 1:500 dilution), Chicken-GFP (AVES, GFP-1020. 1:500 dilution), Rabbit-GABA (Sigma-Aldrich, A2052, 1:500 dilution), Mouse-nc82 (Hybridoma Bank DSHB, Brunchpilot, 1:50 dilution). On the second day, tissues were washed for 4 × 20 min and then incubated in secondary antibodies. The secondary antibodies were all from Invitrogen and used at 1:200 dilution: Alexa Fluor 555 anti-Rabbit (A-21428), Alexa Fluor 488 anti-Mouse (A11001), Alexa Fluor 647 anti-Mouse (A-2123511031). After incubation, tissues were washed for 4 × 10 min. VNC and brains were mounted on a slide for imaging. An Olympus FV1000 microscope with 20X air lens or 40X oil-immersion lens was used for confocal imaging.

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.