Dfc7000 gt camera

Microscopy measurements were carried out on a Leica DMi8 microscope equipped with a Leica DFC7000 GT camera. Dylight® 488-labeled proteins were each mixed with ×200 excess of the same, non-labeled, protein. Phase separation was then induced by changing the pH of the protein solution as described earlier. The solution was incubated at 25 °C and droplets were visualized with ×100 oil-immersion objectives with brightfield, and/or fluorescence microscopy (applying a FITC filter).

Cells were treated with 0.05 μg/ml demecolcine for 2 h before harvesting by trypsinization, resuspended in hypotonic solution (0.056 M KCl), and incubated at 37°C for 7 min. Cells were fixed in cold methanol:glacial acetic acid (3:1) solution overnight at 4°C. Fixed cells were dropped onto glass slides, incubated at 70°C for 1 min in a humidified oven, and air-dried overnight at room temperature. Slides were incubated in 4% formaldehyde in PBS for 5 min, washed three times in 1× PBS and dehydrated with 70%, 95% and 100% ethanol. Hybridization was done with 100 nM Cy3-[CCCTAA]₃ PNA probe (PNA Bio) in 70 μl hybridization mix (10 mM Tris pH 7.4, 70% formamide, 0.5% blocking reagent (Roche)), at 80°C for 3 min, followed by 3 h at room temperature, in a humidified chamber. Slides were washed twice with wash buffer 1 (10 mM Tris pH 7.4, 70% formamide) for 15 min/wash, and then washed three times with wash buffer 2 (0.1 M Tris pH 7.4, 0.15 M NaCl, 0.08% Tween-20) for 5 min/wash. DAPI was added to the second last wash at 0.1 μg/ml. Slides were dehydrated with 70%, 95% and 100% ethanol, air-dried and mounted with Vectashield.
Images were acquired with an Upright Zeiss Axioplan equipped with a 100×/1.40 oil objective, or with a Leica SP8 confocal microscope equipped with a 63×/1.40 oil objective and a DFC 7000 GT camera.

For cohort B, images for quantification were taken using a Leica DMi 8 microscope equipped with a Leica DFC 7000 GT camera. Focus points were set at 20 × magnification at the area of interest, pictures were then imaged and exported as LIF files. Confocal images were taken using Leica TCS SP8, Nikon AX R or Nikon A1 microscopes. All confocal pictures were taken as z-stack images consisting of 10 to 20 layers with a 0.5 to 0.7-μm step size. Heights for z-stacks were identified manually by imaging DAPI.