35 mm petri dish

All functional imaging experiments were performed ex-vivo from brains of 1- to 4-day-old male or female brains on an Ultima two-photon laser scanning microscope (Bruker Nanosystems) equipped with galvanometers driving a Chameleon Ultra II Ti-Sapphire laser. Images were acquired with an Olympus 60x, 0.9 numerical aperture objective at 512 × 512 pixel resolution.
Flies were placed on food containing 400 μM all trans-retinal for 18–36 hr prior to dissection. Brains were dissected in saline (108 mM NaCl, 5 mM KCL, 2 mM CaCl2, 8.2 mM MgCl2, 4 mM NAHCO3, 1 mM NaH2PO4, 5 mM trehalose, 10 mM sucrose, 5 mM HEPES, pH 7.5 with osmolarity adjusted to 275 mOsm), briefly (45 s) treated with collagenase (Sigma #C0130) at 2 mg/mL in saline, washed, and then pinned with fine tungsten wires in a thin Sylgard sheet (World Precision Instruments) in a 35 mm petri dish (Falcon) filled with saline. MBONs were stimulated with 400-500ms of 627 nm LED. For recordings in the LAL (VT018476 and VT055139) ROI were positioned over SMP. For recordings in the FSB (476H09) ROIs were positioned over SMP or FSB.
All image processings were done using FIJI/ImageJ (NIH). Further analysis was performed using custom scripts in ImageJ, Microsoft Excel, and RStudio. Normalized time series of GCaMP fluorescence were aligned to the time point when the opto-stimulus was applied for each replicate.

About 2 mM (proteo)liposomes and 10 µg/ml DAPI (4′,6-diamidino-2-phenylindole; Sigma) (injection marker) in HB150 were filled in Femtotips (Eppendorf); 1 × 10⁴ HeLa cells were plated on poly-l-lysine (Sigma-Aldrich)-coated 12 mm coverslips (Marienfeld GmbH) and then the coverslip was placed into a 35-mm petri dish (Becton Dickinson) filled with pre-warmed injection medium (F12 medium (Invitrogen), supplemented with 10% FCS, 10 mM HEPES (pH7.5), and 100 units/ml each of penicillin and streptomycin). Microinjection was performed using a Femtojet (Eppendorf) and Injectman micromanipulator (Eppendorf) under a Leica DMIL inverted microscope for 5 min per coverslip. After microinjection, the cells were incubated at 37 °C in the cell culture medium and then fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS for 10 min followed by immunocytochemistry using antibodies specific for organelles as indicated. To label Tfn-positive endosomes, microinjection was performed in 5 µg/ml Alexa488-, Alexa568-, or Alexa633-Tfn-containing injection medium for 5 min. After microinjection, the cells were incubated for 5 min at 37 °C in the cell culture medium, then were fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS for 10 min. To take time-lapse images, injected cells were quickly set on an LSM 780 confocal microscope (Carl Zeiss).

Microinjection into HeLa cells was performed as described^{26 (link),27 (link)}. 2 mM lipid (proteo)liposomes, 10 µg/ml DAPI (injection marker) in HB150 were filled in Femtotips (Eppendorf). HeLa cells harvesting 12 mm coverslip was placed into a 35 mm petri dish (Becton Dickinson) filled with pre-warmed culture medium (F12 medium (Invitrogen), supplemented with 10% FCS, 10 mM HEPES (pH7.5) and 100 units/mL each of penicillin and streptomycin). Microinjection was performed using Injectman micromanipulator (Eppendorf) under a Leica DMIL inverted microscope. After microinjection, the cells were incubated for 5 min at 37 °C in the culture medium and then fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 min followed by immunostaining using antibodies specific for organelles as indicated. To label transferrin-positive endosomes, 5 µg/ml Alexa Fluor 488-Transferrin (final concentrations) were added to the culture medium at the beginning of the injections. After microinjection, the cells were incubated for 5 min at 37 °C and then processed for immunostaining as above. For internalization of Alexa Fluor 488-EGF into endosomes, after HeLa cells were starved in DMEM medium (Lonza) for 3 h, microinjection was performed in 100 ng/ml Alexa Fluor 488-EGF containing injection medium for 5 min and incubated for 5 min at 37 °C in the cell culture medium.