Cv1000 system

Securin-GFP cDNA was a gift from Katja Wassmann. The purified linear Securin-GFP DNA was used as template for RNA production. T3 transcription kit (Thermo, AM1348) was used for Securin-GFP RNA production (Niault et al., 2007 (link)). GV oocytes were microinjected with 10 pl of Securin-GFP RNA at 500 ng/μl using an inverted Nikon microscope (Nikon, Japan). Live imaging of oocytes was acquired by a CV1000 system (Yokogawa, Japan) with a 20× objective lens.

Cultures of cerebral walls were prepared as described previously (Saito et al., 2019; Shinoda et al., 2018). Briefly, cerebral walls were microsurgically processed and embedded (ventricular surface top) in a polystyrene cell culture dish (Corning) with Atelocell IAC‐30 collagen gel (Koken) at a concentration of 0.8 mg/ml. Confocal time‐lapse images (en face, parallel to the ventricular surface) were obtained with an upright BX51W1 microscope (Olympus) equipped with a CSU‐X1 laser scanning confocal unit, a 60 × objective lens (LUMFL N 60XW, N.A. = 1.10 Olympus) and an iXon + EMCCD camera (Andor); an on‐stage culture chamber (Tokai Hit) was filled with 45% N₂, 40% O₂, and 5% CO₂ on a CV1000 system (Yokogawa) with a 60× objective lens (PlanApo 60×/1.40 Oil, N.A. = 1.40 Olympus).