Flies were raised at 25°C on a standard cornmeal/molasses diet in bottles or vials, under a 12:12 hr dark:light cycle, and we dissected 3–4 day old female flies (for genotypes see Appendix 2—table 1.). For trans-Tango analyses we dissected 17–18 day old female flies raised at 18°C (Talay et al., 2017).

Immunohistochemical staining was conducted as previously described (Omoto et al., 2017; Omoto et al., 2018). Briefly, brains were dissected in phosphate buffered saline (PBS) and fixed in ice–cold 4% EM–grade paraformaldehyde in PBS for 2.5 hr. They were subsequently washed for 4 × 15 min in ice–cold PBS followed by cold ethanol dehydration (5 min washes in 5, 10, 20, 50, 70, 100% EtOH). After incubation for approximately 12 hr in 100% EtOH at −20°C, brains were subjected to a rehydration procedure with EtOH in the reverse sequence. Brains were then washed for 4 × 15 min in ice–cold PBS and 4 × 15 min in ice-cold 0.3% PBT (PBS with 0.3% Triton X–100), followed by 4 × 15 min in room temperature (RT) 0.3% PBT. They were then incubated in blocking buffer (10% Normal Goat Serum in 0.3% PBT) for 30 min at RT. Following this, the brains were incubated in primary antibodies, diluted in blocking buffer at 4°C for approximately three days. They were subsequently washed 4 × 15 min in RT 0.3% PBT and placed in secondary antibodies diluted in blocking buffer at 4°C for approximately 3 days. They were finally washed 4 × 15 min in RT 0.3% PBT and placed in VectaShield (Vector Laboratories) at 4°C overnight before imaging. trans–Tango and GRASP analyses required separate staining of neuropil and respective fluorophores due to different incubation times.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.