OOCYTE DISSECTION SOLUTION

OR2 Stock (10X, 500 ml)

NaCl 24.09 g

KCl 0.745 g

MgCl2.6H2O 1.017 g

HEPES 5.95 g

Adjust pH to 7.4, bring volume up to 500 ml. Autoclave or filter-sterilize. Store at 4⁰C.

Anasthetise frog

In a 2L container

0.75g of Tricain (-20C freezer)

1.4g Sodium Bicarbonate

up to 500 mL of Milli-Q water

1. Use a bucket full of ice and put the 2L container on ice.

2. Put the frog in the solution for NO MORE THAN 10 MINUTES

3. Procede the surgery

4. After the surgery, empty the container and fill up with TAP WATER up to 400 mL and put the frog back in water

5. Allow at least 2H to the frog to recover before to bringing it back to Animal Facility

6. Try to keep the frog away from light and noise.

Oocytes procedures

1- As soon as the oocytes are extracted put them in 1X OR₂

2- After the surgery, put the oocytes in 7ml of collagenase (Sigma C-0130) solution 1.3mg/ml for 1H shaking at RT. (If the volume of the oocytes + collagenase in the tube is more than 10 ml split the oocytes into two tubes)

3- After 1H, change the collagenase solution like in step two and incubate for 1H shaking RT.

4- Then rinse at least 6X with 1X OR2 solution and put the oocytes in a 60mm dishe full of 1X Barth solution and put the plate in the oocytes incubator.