Tricaine methane

Adult female X. laevis were obtained from Xenopus One (Ann Arbor, MI), housed in an established frog colony, and fed regular frog brittle twice a week. For the removal of oocytes, frogs were anaesthetized by immersion in 0.5% (wt/vol) tricaine methane-sulfonate/ethyl 3-aminobenzoate methanesulfonate salt (MS222; Sigma-Aldrich, St. Louis, MO) until unresponsive to a painful stimulus. A 1-cm incision was made in the abdominal wall through both skin and muscle layers, and then a lobe of ovary containing oocytes was excised. The wound was closed in two layers, and the animal was allowed to recover from anesthesia. Ovarian follicles were removed and digested by gentle agitation in ND96 solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl_2, 1 mM MgCl₂, 10.0 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH 7.4), containing 2 mg/ml collagenase type 1 (Worthington, Lakewood, NJ) twice, each time for ∼60 min. After digestion, healthy stage VI oocytes were manually selected based upon size and uniformity of color. They were then incubated at 18°C in ND96 solution supplemented with 0.1 mg/ml penicillin and 0.05 mg/ml gentamicin sulfate. These procedures were performed under the approved University of Alberta Animal Ethics protocol (#599/06/10/C).