Tsc sp5 inverted confocal microscope

At 24 hpf, embryos were screened for fluorescence, dechorionated and fixed or anesthetised according to the experiment. To image the co-localization of ER_S-β₁₁ and OMM-GFP_1–10 with mitochondria and the ER, fish were fixed for 2 h at RT with 4% PFA, then washed with PBS and mounted in low melting agarose (1.3%, Euroclone) on glass coverslips.
For in vivo imaging, fish were anesthetised and mounted on 35 × 10 mm glass bottom Petri dishes (Ted Pella, INC. Prod. No. 14023-20) in low melting agarose (1.3%, EuroClone). Fish water containing tricaine methanesulfonate 0.61 mM (Sigma) was added in the Petri dishes, in order to keep fish anesthetised. Mounted fish were imaged at RT (20–23 °C) using a Leica TSC SP5 inverted confocal microscope, using either a HCX PL APO ×63/numerical aperture 1.40–0.60 or a HCX PL APO ×100/numerical aperture 1.4 oil-immersion objective. To count ER-mito contacts, a complete z-stack of the cell was acquired every 0.29 µm. To acquire a representative image of a whole fish expressing pT2-DsRed-UAS-SPLICS_S-P2A, a ×10 HCPX PL Fluotar NA 0.3 objective was used.

To selectively express the SPLICS probe in zebrafish neurons, the Gateway technology was exploited to generate the pT2–DsRed–UAS–SPLICS vector [31 (link)], which allows the simultaneous expression of both a cytosolic dsRED to mark neurons and the SPLICS probe. The resulting plasmid was injected into 1–2 cell stage s1102t:Gal4 embryos (either WT or psen2^−/− embryos in this transgenic background) at a concentration of 50–60 ng/µL. At 24-hpf, embryos were screened for fluorescence, dechorionated and anesthetized. Fish were then mounted on µ-Slide 8-Well Glass Bottom (Ibidi Gmbh, Munich, Germany; Cat# 80,827) in 300 µL/well low-melting agarose (1.2%, EuroClone, Milan, Italy). Fish water containing tricaine methanesulfonate 0.61 mM (Merck KGaA, Darmstadt, Germany) was added above each well, in order to keep fish anesthetized. Mounted fish were imaged at RT (20–23 °C) using a Leica TSC SP5 inverted confocal microscope, using either an HCX PL APO ×63/numerical aperture 1.40–0.60 or an HCX PL APO 100X/numerical aperture 1.4 oil-immersion objective. To count ER-mitochondria contacts, a complete Z-stack of the cell was acquired every 0.5–0.7 μm.

All animal experiments were conducted on wild-type fish. Adult fish were maintained and raised in 5 l tanks with freshwater at 28 °C with a 12 h light/12 h dark cycle. Embryos were obtained from spontaneous spawnings and raised at 28 °C in Petri dishes containing fish water^{92 (link)}. To perform experiments, both wt and s1102t:GAL4 fish were used. All experiments were conducted on 24 h post fertilization (hpf) embryos. The pT2-DsRed-UAS-SPLICS_S-P2A vector has been already described^{35 (link)}. Before injections, all plasmids were diluted in Danieau solution (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO₄, 0.6 mM Ca(NO₃)₂, 5 mM HEPES pH 7.6) and 0.5% phenol red. At 24 hpf, embryos were screened for fluorescence, dechorionated, and anesthetised with tricaine. They were then mounted on 35 × 10 mm glass bottom Petri dishes (Ted Pella, INC. Prod. No 14023-20) in low melting agarose (1.3%, EuroClone). Fish water containing tricaine methanesulfonate 0.61 mM (Sigma) was added in the Petri dishes, in order to keep fish anesthetised. Mounted fish were imaged at RT (20–23 °C) using a Leica TSC SP5 inverted confocal microscope, using a HCX PL APO 63X/numerical aperture 1.40-0.60 oil-immersion objective. To image ly–mt contacts, a Z-stack of the cell was acquired.

The human WT, A53T or A30P mutant αsynS₁₁ plasmids were injected into 1 cell stage WT eggs together with a plasmid expressing OMM-GFP_1–10, IMS-GFP_1–10 or mt-GFP_1–10. For injections, all plasmids were diluted in Danieau solution (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4, 0.6 mM Ca (NO3)2, 5 mM HEPES pH 7.6) and 0.5% phenol red. For the injections, the concentration of each plasmid was 50 ng/ul. At 1dpf, about 30 embryos were screened for fluorescence, dechorionated and anesthetized. For in vivo imaging, about one third of the injected embryos showed fluorescent signal and half were anesthetised and mounted on 35 × 10 mm glass bottom Petri dishes (Ted Pella, INC. Prod. No. 14023-20) in low melting agarose (1.3%, Euro- Clone). Fish water containing tricaine methanesulfonate 0.61 mM (Sigma) was added in the Petri dishes, in order to keep fish anesthetized. Mounted fish were imaged at RT (20–23 °C) using a Leica TSC SP5 inverted confocal microscope, using a HCX PL APO ×63/numerical aperture 1.40–0.60. To image reconstituted GFP, a complete z-stack of the cell was acquired every 0.29 μm and shown as Z-projection of several planes.