Preparation and Microinjection of Xenopus Oocytes

1. Female Xenopus laevis frogs (Nasco, Fort Atkinson, WI; see Notes 1-3).

2. MS-222 (Sigma, St. Louis, MO). Store at -20°C in a desiccator. This is a possible carcinogen; wear gloves.

3. Frog guillotine.

4. Gentamicin solution, 50 mg/mL (Sigma). Store at 4°C; toxic.

5. Modified Barth's solution (MBS): 88 mMNaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mMMgSO4, 0.33 mM Ca(NO3)2, 0.14 mM CaCl2, 5 mM N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid (HEPES), pH 7.4, containing 0.5 mg/mL gentamicin. Store at 17°C (see Note 4).

6. Two forceps (Dumont 5) (Fine Science Tools Inc., Foster City, CA).

7. Upright light stereo dissection microscope.

8. Regular Petri dishes and one lined with nylon netting (for oocyte injection).

10. Collagenase type 1 (Sigma). This is toxic.

11. Ringer's solution: 120 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 5 mM HEPES, pH 7.3. Store at 4°C (see Note 4).

12. Glass capillaries (7-in. Drummond 3-00-203-G/XL).

13. Horizontal pipet puller (Narishige, Tokyo, Japan).

14. Microinjector (Drummond Nanoject Drummond Scientific Co, Broomall, PA).

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