Fv1200 confocal microscope

Adult age-matched male flies, following 7 days of CD or SD, were fasted on a wet Kimwipe for 18–24 hr before prepping for in vivo confocal laser imaging. As previously described (May et al., 2019 (link); LeDue et al., 2015 (link)), the preparation consisted of a fly affixed to a 3D-printed slide with melted wax around the head and on the dorsal part of the thorax. Distal tarsal segments were removed to prevent interference of the proboscis stimulus, and the proboscis was wax-fixed fully extended with the labellum functional and clear of wax so that proboscis contraction and extension could not perturb the brain’s position. A glass coverslip was placed such that artificial hemolymph (108 mM NaCl, 8.2 mM MgCl₂, 4 mM NaHCO₃, 1 mM NaH₂PO₄, 2 mM CaCl₂, 5 mM KCl, 5 mM HEPES) placed over the head did not touch the proboscis. Data were acquired with a FV1200 Olympus confocal microscope, a 20x water immersion objective, and a rate of 0.254 s per frame. Stimuli consisted of a brief touch of a small Kimwipe soaked in milliQ water or 30% sucrose solution to the labellum. Responses to both sucrose and water were measured in the same fly. For calcium imaging experiments, n counts each ROI, of which there are two per fly.

Immunofluorescence were performed in TGF-β1-treated ESCs to detect the co-localization of GLS1 and α-SMA. ESCs were seeded on 35 mm confocal dishes (Cellvis) at a density of 1 × 10⁴ cells/mL. After treatments, cells were fixed with 4% PFA for 15 min, permeabilized with 0.2% Triton X for 10 min and then blocked with 5% BSA for 45 min at room temperature. Primary antibodies α-SMA (1:200; Abcam, ab7818), GLS1 (1:200; Abcam, ab93434) were used for overnight incubation at 4 °C. DyLight488-conjugated goat anti-rabbit antibody (1:200; Multisciences, GAR4882) and DyLight594-conjugated goat anti-mouse antibody (1:200; Multisciences, GAM5942) were used for detection. DAPI was used to stain cell nucleus, and the stained ESCs were observed by an Olympus FV1200 confocal microscope (Olympus).

Following different interventions for 24 h, the F11 cells were fixed by 4% paraformaldehyde and permeabilized by 0.1% TritonX-100 (Sigma Aldrich) for 10 min. Unspecific bindings were blocked using 8% bovine albumin at room temperature for 1 hour. Cells were incubated with primary antibody TUBB3 (1:100, Abcam) overnight at 4°C. The next day, the cells were incubated with goat anti-mouse antibody (Alexa Fluor 488, 1:500, Abcam). DAPI was used to visualize cell nucleus. The cells were detected under the Olympus FV1200 confocal microscope (Olympus). The number and length of neurites were calculated by Image J from five separated high-power fields in each group. Each experiment was performed three times.

Immunofluorescence was conducted as described in our previous research (Peng et al., 2019 (link)). Specifically, the cells were fixed in 4% paraformaldehyde for 30 min and then treated with 0.3% Triton X-100 for 5 min. For non-specific blocking, the cells were incubated in 0.2% bovine serum albumin (Calbiochem, San Diego, CA, USA) for 15 min and then incubated with anti-α-tubulin antibody (dilution ratio was 1:100; GeneTex, Inc., North America, GTX628802) at 4°C for 12 h. The cells were stained with anti-mouse IgG fluorescent secondary antibody (dilution ratio was 1:200; Abways Biotechnology Co., Ltd., Shanghai, China, AB0132) for 2 h at room temperature. DNA was stained with Hoechst 33342 (Invitrogen). Fluorescence was imaged using OLYMPUS FV1200 confocal microscope (Olympus, Tokyo, Japan).

Male flies harboring the Venus-tagged dopamine receptors^{52 (link)} were presented with 5% sucrose solution (“Sugar only”) or 5% sucrose solution and 5% sucrose solution supplemented with 15% ethanol (“With ethanol”) in the CAFE assay chamber for 3 days. Fly brains were then dissected in PBS, immediately fixed in 2% paraformaldehyde in PBS for 1 h at room temperature, and washed three times with PBST (0.1% Triton X-100 in PBS). The brains were then blocked with 3% goat serum in PBST for 30 min at room temperature and incubated in the primary antibody solution (rabbit anti-GFP (1:1000; catalog #A11122, Thermo Fisher Scientific, MA, USA) and 1% goat serum in PBST) and in the secondary antibody solution (Alexa Fluor 488 goat antirabbit (1:1000; catalog #A11034, Thermo Fisher Scientific) and 1% goat serum in PBST) at 4 °C for two nights respectively. Brains were then washed for three times with PBST and mounted in SeeDB2G^{77 (link)}. Images were obtained using the Olympus FV1200 confocal microscope (Olympus, Tokyo, Japan) with the 20x, 0.85NA oil objective lens (UPLSAPO20XO, Olympus). Images of the two groups were acquired at the same time periods under the identical microscope settings.

LysoTracker Red DND-26 (Invitrogen, L7528) was diluted in M9 and dispensed on NGM dishes, M9, or CeMM to yield a final concentration of 1 μM. Worms were cultured on these dishes or liquid solutions in the dark and transferred to NGM dishes or fresh liquid without dye for 30 min. The animals were paralyzed with 25 mM levamisole (Sigma-Aldrich, 196142) in M9, mounted on 3% agarose pads on microscope slides, and imaged with an Olympus FV1200 Confocal Microscope (Olympus, Tokyo, Japan).