Airyscan processing tool

Dechorionated embryos mounted in a glass-slide with a gas permeable membrane (Tsarouhas et al., 2007 (link)). Widefield live imaging performed to analyze protein clearance and gas-filling on embryos as described in Tsarouhas et al., 2019 (link). For confocal live imaging, embryos were imaged with a scanning confocal microscope (LSM 780, or 800 Carl Zeiss) equipped with an Argon and an HeNe 633 laser using a C-Apochromat ×63/1.2 NA water objective. Z-stacks with a step size of 0.5–1.0 μm were taken every 6 min over a 3–8 hr period. For high-resolution confocal live imaging, an Airy-scan-equipped confocal microscope system (Zeiss LSM 800, Carl Zeiss) was used. Z-stacks (0.16–0.2 μm step size) were taken every 15 min over a 2–4 hr period using a Plan-Apo 63 ×/1.40 DIC oil or a C-Apochromat ×63/1.2 NA water objective (Zeiss). Raw data were processed with the Airy-scan processing tool available on the Zen Black software version 2.3 (Carl Zeiss). Images were converted to tiff format using the Zen Black or ImageJ/Fiji software.

Cells transfected with eGFP-ATG9B or eGFP-ATG9B/mCherry-ATG9A were seeded on glass-bottom microwell dishes (MatTek Corp., P35G-1.5-14-C) to reach 70% confluency the day of the experiment. Cells were imaged using a Zeiss LSM 880 Airyscan confocal microscope with Plan-Apochromat 63×/1.4 Oil DIC M27 objective lens. Live imaging was performed using Zeiss ZEN imaging software and the acquired movies were processed using an Airyscan processing tool on the ZEN software provided by Zeiss. Plasmids used in this study are listed in Table S1.

Cells were grown on poly-D-lysine treated coverslips to reach 70% confluency the day of the experiments. After the treatments, cells were fixed with 4% paraformaldehyde in PBS supplemented with 0.1 mM CaCl₂, 0.1 mM MgCl₂ for 10 min at room temperature. Cells were washed three times with PBS before adding 50 mM NH₄Cl for 10 min at room temperature and then permeabilized with 50 μg/mL digitonin (Merck Millipore; D141) for 5 min at room temperature. Cells were then washed with PBS and blocked in 5% BSA (in PBS for 30 min at room temperature. Coverslips were incubated upside down with primary antibody in 1% BSA in PBS 1 h at room temperature, then washed three times with PBS and incubated upside down with secondary antibody in 1% BSA in PBS for 1 h. Finally, coverslips were washed three times in PBS and once with deionized water before mounting them on glass microscope slides using 10 µL Mowiol mounting solution (Millipore,475904). Fluorescence images were acquired using a Zeiss LSM 880 Airyscan confocal microscope with Plan-Apochromat 63×/1.4 Oil DIC M27 objective lens. Zeiss ZEN imaging software was used to acquire the images, and, after acquisition, processing was performed using an Airyscan processing tool on the ZEN software provided by Zeiss. All the antibodies used in this study are reported with the corresponding working concentrations in Table S1.