Zoolog Sci. 2004 Feb;21(2):153-7 doi:10.2108/zsj.21.153

Shoguchi E1, Ikuta T, Yoshizaki F, Satou Y, Satoh N, Asano K, Saiga H, Nishikata T.

Fluorescent in situ hybridization to ascidian chromosomes.

“Embryos at three different developmental stages (32-cell embryos, 64-cell embryos and gastrulae) were used to produce metaphase spreads, although treatment of 32-cell embryos and 64-cell embryos with 0.05% colchicine (Sigma) in seawater caused aggregated and disorganized chromosomes. After treatment with colchicine for 20–30 min, embryos were transferred to a 1.5-ml microfuge tube, to which cold methanol:glacial acetic acid (3:1) fixative was added (chilled on ice). The fixative was changed twice over a period of 1 hr and once after overnight (embryos kept at 4°C). The specimens were then stored in fixative at 4°C.”