Preparation of Oocytes

3.2.1. Anesthesia of Frog

A female frog is anesthetized in a 2000-mL container containing a mixture of 200 mL 2X benzocaine buffer and 200 mL ice for about 10 to 15 min (see Note 3).

3.2.2. Dissection and Suturing

Place the frog on her back on an ice bed. Using a sterile scalpel, make an incision about 0.8 to 1.0 cm wide at the abdomen in two steps (see Note 4) (Fig. 4). First, using evenly distributed pressure, carefully incise the skin. Second, lift the skin off the muscle layer using a pair of forceps and make a small and precise incision through the muscle layer. Take out the ovarian lobes carefully, overestimating the number of oocytes needed, and place them in 1X MBS buffer. Suture the muscle layer first with a few stitches and then suture the skin. Monitor the frog closely for a day to ensure that she is healthy before returning her to the tank.

3.2.3. Separation/Isolation of Oocytes (see Note 5)

1. Place the oocytes in a Petri dish filled with 50 mL of OR2 buffer.

2. Cut the membrane of the ovarian lobes.

3. Swirl the dish slowly for 10 min, changing the buffer five times.

4. Change OR2 buffer to collagenase type I buffer.

5. Swirl gently for 20 min on a shaker (see Note 6).

6. Remove healthy individual oocytes to new OR2 buffer in a new Petri dish.

7. Swirl and change the buffer twice.

8. Change OR2 buffer to 1X MBS buffer containing gentamicin.

9. Keep the healthy oocytes at 18°C (see Note 7).

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