Tsc sp2

To image Drosophila da neurons at the wandering third instar (wL3) or WP stage, larvae or pupae were first washed in PBS buffer briefly, followed by immersion with 90% glycerol. For imaging da neurons at 16 h APF, pupal cases were carefully removed before they were mounted with 90% glycerol. Dendrite images were acquired on Leica TSC SP2. Subcellular localization images were acquired on Leica TCS SP8 STED 3× super-resolution microscope.

Eyes were isolated from adult mice treated with 4 or 10 weeks of tamoxifen as described or from 10-day-old pups that were fed tamoxifen from P2 to P6. Eyes were fixed in 4% PFA and then the retinas dissected out. For isolectin B4 staining, retinas were permeabilized in 0.5% Triton X-100/1% BSA/PBS for 2 h. For all staining, retinas were incubated with primary antibodies anti-CD31 (1:50), anti-collagen type IV (1:100, Abcam), anti-GFP (1:200), anti-SMA (1:100) or isolectin B4 (1:50) followed by secondary antibodies at 1:1,000 dilution. Staining was visualized using confocal microscopy (Leica TSC SP2).

Transfected cells were plated on sterile 15‐mm‐diameter coverslips (Thermo Fisher Scientific) coated before cell plating with Poly‐L‐Lys for 20 minutes at RT (Sigma‐Aldrich). After adhesion, the cells were fixed in ice‐cold 100% methanol (Sigma‐Aldrich) for 5 minutes. The coverslip was mounted upside down on a microscope slide using a drop of mounting medium composed by 50% PBS 2×, 50% glycerol 100% and 1% m/v n‐propyl gallate; pH = 8. Confocal images were obtained with a confocal laser‐scanning fluorescence microscope (Leica TSC‐SP2).

The cultured cells were seeded onto six-well plates, and their differentiation potency was characterized and tested using immunofluorescence staining analysis. To fix the cells, 4% paraformaldehyde (PFA) was used. Mouse monoclonal anti-nestin antibody (1:500, Abcam, Boston, MA, USA), rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) antibody (1:500, Abcam), and mouse monoclonal anti-β-tubulin III antibody (1:500, Abcam) were used for NPCs identification. D4′, 6′-diamidion-2′-phenylindole (DAPI, 1:1000, Abcam) was used to counterstain the nuclei. To detect CypD and Complex V (mitochondrial marker), anti-CypD (1:200, ab110324, Abcam) and anti-complex V (1:200, 45, 9000, Invitrogen, Carlsbad, CA, USA) antibodies were used. After fixing the cells for 20 min, they were placed in 0.1% Triton X-100 for 30 min, followed by a blocking buffer (10% goat serum) for 1 h. The cells were then incubated with primary antibodies overnight at 4 °C. Samples were incubated in secondary antibodies [fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC)-conjugated IgG] for 2 h, followed by DAPI incubation for 5 min at room temperature. The slides were rinsed with DPBS, and the images were captured using a laser confocal microscope (TSC SP2, Leica, Manheim, Germany).

To image Drosophila da neurons at the wandering 3^rd instar (wL3) or WP stage, larvae or pupae were first washed in PBS buffer briefly and followed by immersion with 90% glycerol. For imaging da neurons at 16 hr APF or 20 hr APF, pupal cases were carefully removed before mounted with 90% glycerol. Dendrite images were acquired on Leica TSC SP2.

Rat kidneys were fixed overnight with 4% paraformaldehyde at 4°C, cryopreserved in 30% sucrose for 24 hours, and embedded in optimal cutting temperature medium. Thin transverse cryosections (4 μm) were placed on Superfrost/Plus Microscope Slides (Thermo Fisher Scientific). The sections were incubated with a rabbit polyclonal antibody directed to AQP2 (1:1000 dilution)23 and then with an AlexaFluor488‐conjugated secondary antibody (Life Technologies, Carlsbad, CA, USA). Confocal images were obtained with a confocal laser‐scanning microscope (TSC‐SP2; Leica, Wetzlar, Germany).