Imaging spacer

To increase contrast during fluorescence imaging, Cy5 (free or RNA-labelled), Alexa Fluor 488 (Alexa 488; Thermo Fisher Scientific) or mCitrine (YFP), were included at final concentrations of 1 μM, 0.6 μM and 2 μM, respectively. Images were captured at room temperature using a Leica TCS-SP5 confocal fluorescence microscope and an HCX PL APO CS 63x (NA 1.40) oil immersion objective. Samples were illuminated with 488 nm (Alexa 488), 514 nm (YFP), or 633 nm (Cy5) lasers, with power and gain settings adjusted to give mean fluorescence intensity values of protein condensates of approximately 50–70% of the maximum 16-bit depth (∼32,000–46,000 a.u.). Images were typically captured with settings of 256 × 256 pixels at ∼ 98 × 98 × 98 nm (XYZ) resolution, 1400 Hz scan speed and a line average of 2.
Phase separation was initiated by mixing protein solutions with a buffer of lower ionic strength, as described above, and were left for 10 min to promote droplet growth. Phase separated solutions were then transferred to a 0.22 mm thick siliconized glass coverslip (Hampton Research), before sealing with 0.12 mm imaging spacers (Sigma) and a second siliconized glass coverslip. Samples were then left to equilibrate for approximately 50 min prior to imaging.

After zygote collection, the cells were fixed in 4% PFA for 30 min, before permeabilization in 0.2% Triton X‐100/PBS (PBSTX) for 30 min. Cells were then blocked in 10% goat serum (Dako) in PBSTX either at 4°C overnight or for several hours at 4°C followed by incubation at room temperature. Cells were incubated overnight at 4°C in primary antibody (anti‐Scc1, Millipore #05‐908, 1:250). After washing in blocking solution for at least 30 min, incubation with the secondary antibody (anti‐mouse IgG (H + L), Thermo Fisher Scientific #A‐11001, 1:500) was carried out for 1 h at room temperature. Another set of washing steps in 0.2% PBSTX was followed by a quick PBS wash and mounting of the cells in Vectashield containing DAPI (Vector Labs) using imaging spacers (Sigma‐Aldrich). In situ fixed zygotes were imaged on a confocal microscope (LSM780, Zeiss, ZEN black) using a 63×, 1.4NA oil objective. The presence of DNA compaction reminiscent of vermicelli in Wapl zygotes was classified using ImageJ and 3D visualization by Imaris (8.1.2). Brightness and contrast of images presented were adjusted using ImageJ software. No blinding or randomization was used for handling of the cells. Samples were excluded from the analysis if cells were not fertilized or in the wrong cell cycle phase (PN stage).