Immunostaining of Larval Neurons

Dissected larvae were processed as previously described (Mukherjee et al., 2020 (link)). Briefly, fillet preparations were fixed in freshly prepared 4% formaldehyde for 20 min at room temperature and were then washed four times for 10 min in PBS plus 0.1% Triton X-100 (PBST). Blocking was carried out in PBST plus 5% BSA for 1 h at room temperature. Preparations were incubated with appropriate primary antibodies diluted in PBST overnight at 4°C. After washing in PBST for ∼8 h, changing washes every 30–45 min, samples were incubated in secondary antibodies diluted in PBST overnight at 4°C. The fillet preparations were then washed for ∼8 h, changing washes every 30–45 min in PBST before mounting in Mowiol. They were stored at −20°C and imaged within a week. Neurons within segments A2 to A6 were imaged. Imaging of γ-tubulin–GFP within the soma was carried out using an Olympus FV3000 scanning inverted confocal system run by FV-OSR software with a 60×1.4NA silicone immersion lens (UPLSAPO60xSilicone). Images of neuronal somas within IT.Gal4-dgt5>UAS-IVS-myr-GFP flies were collected on a Zeiss Axio Observer.Z1 inverted CSU-X1 Yokogowa spinning disk system with 2 ORCA Fusion camera (Hamamatsu) run by Zeiss Zen2 acquisition software using a 60×1.4NA oil immersion lens (Zeiss). Z-stacks with 0.5 µm spacing were acquired to cover all neuronal soma.