Stellaris 5 high resolution laser confocal fluorescence microscope

Cells were seeded on poly-L-lysine−coated coverslips (2.5 × 10⁵ cells) one day prior to the experiment. When ∼ 50% confluency was reached, cells were transiently transfected with 1 µg FUS-GA, 1 µg FUS-B, and 1 µg TDP-43-RA via 6 µl lipofection (X-TremeGene 9, Roche) in 300 µl Opti-MEM^TM (Life Technologies), or 1 µg tandem plasmid CMV-FUS-GA-IRES-FUS-B-IRES-TDP-43-RA via 2 µl lipofection in 100 µl Opti-MEM^TM. Proteins were transiently expressed for 24 h at 37 °C under 5% CO₂ in a HERAcell Vios 160i LK CO₂ incubator (Thermo Fisher Scientific). Cells were treated with 25 µM AdOx (Selleck Chem) for 24 h and stained with 1 µM Hoechst 33342 (Enzo) for 10 min before confocal imaging. Cells transfected with 1 µg TDP-43-GA and 1 µg TDP-43-B or 1 µg TDP-43-eGFP only were treated with 5 µM CuET (TCI) for 3 h before imaging. All confocal images of cells were got with Leica Stellaris 5 High-resolution Laser Confocal Fluorescence Microscope.

Proteins NTD-RBD-GA, NTD-RBD-B, and NTD-RBD-RA were stored at high concentration salt solution (20 mM HEPES, 300 mM NaCl, 1 mM DTT, pH = 7.5). First, NTD-RBD-GA/NTD-RBD-RA and NTD-RBD-B were mixed together in tube 1. Then mixed dilution buffer using low salt solution (20 mM HEPES, pH = 7.5) with 50% PEG 3350 stock solution was added in tube 2. Finally, all the solution in tube 2 was added to tube 1 and mixed well. NTD-RBD- proteins were in phase separation in the final condition: 20 mM HEPES, 140 mM NaCl, 5% PEG 3350; the total protein final concentration was 50 μM. The ratio of NTD-RBD-GA and NTD-RBD-B was chan­ged to get different fluorescent intensity droplets. After all the samples were prepared, 10 μL sample was added to the slide with a spacer, covered with a coverslip, and then turned over and left in an upright position for 10 min, so that the droplets could be adsorbed on the surface of the coverslip.
For FUS-GA/B/RA proteins, TEV protease was added to induce phase separation. The final total protein concentration was 10 μM, and the final LLPS condition was 20 mM HEPES, 200 mM NaCl, 10 µM TEV, 5% PEG 3350. The slides were left in an upright position for 30 min before confocal imaging with Leica Stellaris 5 High-resolution Laser Confocal Fluorescence Microscope with a 63 × oil objective lens.