Nis elements confocal 5

Protoplast images were obtained using a Leica TCS SP8 confocal microscope equipped with a HC PL APO 20x/0.75 IMM CORR CS2 objective (Leica Microsystems, Mannheim, Germany), with the exception of images in Supplementary Fig. 7b and Fig. 5a that were taken with a Zeiss LSM880 Airyscan using a 63x/1.4 oil immersion lens. GFP was excited with a White Light Laser (WLL) at 488 nm and the emission detected at 500–550 nm. YFP was excited with a 514 nm laser line and detected at 520–555 nm. mCherry fluorescence was excited at 561 nm and emission was detected between 575–630 nm. Samples, co-expressing two fluorophores were imaged in sequential mode between frames. All images were taken as z-stacks (internal distance is 0.5 um) and further analyses and projections were performed with either ImageJ/(Fiji) software^{88 (link)} or Imaris.
For GUV experiments: Images were acquired by confocal fluorescence microscopy (Nikon Eclipse Ti-E inverted microscope using a Nikon A1R confocal laser scanning system with laser lines: 405 nm, 488 nm, 561 nm, 640 nm; 60x/1.49 oil immersion objective; Nikon Instruments, Inc.) and analyzed using the corresponding NIS Elements software (NIS Elements Confocal 5.20, Nikon Instruments, Inc.) and ImageJ (Fiji win64, Open source).

The GUVs were formed by a classical electro-formation protocol, as previously described [31 ]. In brief, solutions of lipids at a concentration of 0.5 mg/mL were composed of DOPC, cholesterol, Atto 647N DOPE and the glycosphingolipid C8-LacCer, at a molar ratio (mol-%) of 29.7:30:0.3:40 or Gb3 at 64.7:30:0.3:5. Solutions were prepared in chloroform and spread on indium tin oxide (ITO) covered glass slides. For removing the residual solvent, the slides were incubated under vacuum for at least 1 h. A chamber was assembled with two slides, filled with 318 mOsm·L⁻¹ sucrose solution as formation buffer and an AC electrical field with a voltage of 1 V·mm⁻¹ was applied (to the chamber) for 2.5 h at room temperature. The LgtC reaction on 40 mol-% LacCer GUVs was initiated in 20 mM HEPES, pH 7.4, 1 mM MnCl₂, 1 mM DTT, 100 ng/μL LgtC (2.9 µM), 0.5 mM UDP-Gal and incubated for 1 h. GUVs were observed in hand-built chambers using 318 mOsm·L⁻¹ PBS as an imaging buffer. Images were acquired by confocal fluorescence microscopy (Nikon Eclipse Ti-E inverted microscope using a Nikon A1R confocal laser scanning system with laser lines: 405 nm, 488 nm, 561 nm and 640 nm; 60 oil immersion objective, NA = 1.49; Nikon Instruments, Inc., Melville, NY, USA) and analyzed using NIS Elements software (NIS Elements Confocal 5.20, Nikon Instruments, Inc.) and ImageJ (Fiji win64, Open source).