Embryo Mounting and Cortex Ablation

Embryo preparation and mounting has been described elsewhere (Pohl and Bao, 2010 (link); Dutta et al., 2015 (link)). Mounting was modified by using differently sized polystyrene (15 µm, 20 µm, 25 µm; Polysciences, Hirschberg, Germany) and polymethylmethacrylate spheres (12 µm and 13.5 µm, PolyAn, Berlin, Germany). Microscopy was performed with a VisiScope spinning disk confocal microscope system (Visitron Systems, Puchheim, Germany) based on a Leica DMI6000B inverted microscope, a Yokogawa CSU X1 scan head, and a Hamamatsu ImagEM EM-CCD. All acquisitions were performed at 21–23°C using a Leica HC PL APO 63×/1.4-0.6 oil objective. Cell cortex ablations were performed using a pulsed 355 nm UV laser mounted on the same microscope. One ablation cycle was performed per acquisition with a residence time per pixel of 3.5 ms. Acquisitions pre and post ablation were performed at 200-ms intervals.