2 orca fusion camera

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.

All imaging was performed at RT (∼20°C). Confocal imaging of fixed embryos—and the movies of scaffolds organizing microtubules—was performed on an Olympus FV3000 scanning inverted confocal system run by FV-OSR (Olympus Super Resolution) software using a 60× 1.4 NA silicone immersion lens (UPLSAPO60xSilicone) or 30× 0.95 NA silicone immersion lens (UPLSAPO30xSilicone). Confocal imaging of scaffolds recruiting γ-TuRC proteins or organizing microtubules as well as of testes samples was performed 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× 1.4 NA oil immersion lens (Zeiss). Confocal imaging of oocytes was performed on a Confocal LSM780 mount on a Axio Observer Z1 microscope (Zeiss), detection was done with spectral channels GaAsP, controlled by Zen software using a 25× 0.8 NA plan apochromat oil objective. Phase-contrast microscopy of round spermatids was performed on a Leica DM IL LED inverted microscope controlled by μManager software and coupled to a RetigaR1 monochrome camera (QImaging) using a 40× 0.55 NA air objective (Leica). Movies of scaffolds organising microtubules and spindle-like structures were recorded at a rate of 1 frame per 30 s.

Before imaging, samples were mounted on ProLong (P36962, Invitrogen). Imaging was performed on a microscope setup based on an inverted Axio Observer.Z1 microscope (Zeiss), a Yokogawa CSU-X1 spinning disk, and a 2 ORCA Fusion camera (Hamamatsu). ZEN 2 acquisition software was used for setup-control and data acquisition. Illumination was performed using different lasers (405 nm, 488 nm, 561 nm). Cells were inspected with a 63 × 1.4 oil immersion objective (Zeiss). Images were taken in z-stack focal-planes with distances of 500 nm for a maximal intensity projection.