Xenopus laevis

Updated 10/03/2024

Frog Surgery Prep

Before beginning check that the surgical tools have been autoclaved and expect to spend 4-5 hours to complete the entire protocol.

Frog Anesthetic:

1. Collect the plastic pitcher and fill with 1000 mL of dH2O

2. Add a stir bar and place on the stir plate

3. Weigh 1.5g of Tricaine powder and add to the water

4. Weigh 0.2g of Sodium Bicarbonate and add to water 

*Use clean weigh paper for each powder and make sure that the scale is tared and balanced (the bubble in the left corner should be centered.)*

5. After the solutes are dissolved, remove the stir bar with the magnetic remover

6. Rinse the stir bar and remover and put away

*To euthanize the frog (after surgery #5) weigh an additional 3.5g of Tricaine which will be added to the anesthetic after the anesthesia and surgery have been performed. Put the frog into the euthanasia solution after suturing and leave for 2 hours. Remove head, place carcass in baggie, and put in animal waste barrel in the 3rd floor cold storage room.*

Frog Selection and Anesthesia:

1. Take the anesthesia solution and the square container to the 7th floor

2. Get the black frog book and find which frog is up for surgery and the ID pictures

3. Fill the recovery tank with around 2-3 inches of water

4. Separate the frog using the blue net and place in tank

5. Confirm that the correct frog has been separated and place in anesthesia

6. Cover the pitcher with the square container to prevent the frog from escaping

7. Wait 40 min for frog to go under

Surgery Cart Prep: (do while frog is being anesthetized)

1. Clean the black cart with 70% isopropyl alcohol

2. Place the surgery instruments on the cart in their glass dish

3. Check that there are paper towels in the plastic container at the bottom

4. Check that there is sufficient Frog H2O

5. Collect PGA & Nylon suture from the supply drawer

6. Put a 60 mm petri dish on the cart (fill with DS solution right before surgery)

7. Collect 0.2% lidocaine and plastic cutting board out of respective freezers right before surgery

8. Get liberase from -80° freezer and put in freezer Alice

Surgery:

1. Place three layers of 2 paper towels on the board and wet with Frog water

2. Remove frog from Tricaine and put it on its back on the board

3. Pour some Tricaine into the squirt bottle for later use and set pitcher aside

4. Check the frogs unconsciousness by performing a toe pinch: spread out their toes and pinch the webbing in between, if their breathing is elevated, return to anesthesia till toe pinch elicits no increase in respiration

5. Rinse the frog with water to wet their skin and cleanse the surgery site of Tricaine solution

*Continue rinsing throughout surgery to keep the skin moist.*

6. Make a small incision in her lower abdomen (either L/R) cutting through both skin and muscle

7. Remove eggs using curved tweezers and place in 60 mm DS dish

8. Continue collection until 4-5 sacs are extracted or the dish is mostly full

9. Suture the muscle closed with 3±1 PGA stitches ensuring no oocytes are trapped in the suture

10. Suture the skin closed with 3±1 Nylon stitches

*Use a 3 throw surgeons knot followed by two 2 throw surgeons knots per stitch.*

11. Place lidocaine over wound for 30 secs

12. Put frog in recovery tank on plastic ramp (frog should be mostly submerged with her nose out)

13. Fill out the “Laboratory Animal Surgery/Anesthesia Record” sheet and leave with the frog at the recovery tank

14. Fill out the surgery log in black book and put away

15. Throw away the paper towels and suture bits and go back to 5th floor

16. Dispose of sharps in appropriate container

17. Clean surgical tools with brush and tap water then rinse with dH2O

18. Clean cutting board with tap water and soap then rinse with dH2O

19. Clean cart with 70% isopropyl alcohol and put remaining supplies away

Oocyte Prep:

1. Clean microscope platform and surrounding area with 70% isopropyl alcohol

2. Pull apart the oocytes into small clumps of 4-5 eggs using tweezers

3. Fill 50 mL conical tube with 10 ml of DS

4. Cut off end of large transfer pipet

5. Place separated oocytes into conical tube using pipet and add thawed liberase solution (1 mg/ml) The liberase solution is made by adding 5 ml of sterile water to the bottle containing 5 mg of liberase (Fisher Scientific Cat No. 50-100-3280). Aliquot 200 ul into each 1.5 ml  sterile microcentrifuge tube.

6. Label the tube with the time and contents (frog oocytes + liberase)

7. Place on Nutator rocker for 2 hours at room temperature

Sorting Oocytes:

1. Cut off the end of a small transfer pipet and cut two 1 cm wide strips of ParaFilm

2. Fill two 60 mm petri dishes with DS solution, one with MBSH solution, and a 35mm dish with MBSH+AB+AF solution

3. Transfer the digested oocytes into one of the DS dishes and wrap the edge with ParaFilm

4. Swirl on the Scilogex machine at 100 for 30 sec to 1 min

5. Unwrap and transfer oocytes to the 2nd DS dish, wrap with ParaFilm and swirl

6. If the solution is clear move to step 7, if cloudy repeat step 5

7. Using the small transfer pipet collect good oocytes and transfer to the MBSH dish

*Good oocytes will have an obvious, symmetrical border between the green and brown side; will be perfectly round; have little to no “blemishes” (dark spots, lines, etc..); no bulges; and be similarly sized to one another.*

8. Double check the oocytes in the MBSH solution and if good transfer to 35mm dish

*Discard bad oocytes on lid of DS dish*

9. Label dish with “MBSH-AB-AF,” Frog Letter, date, and initials

10. Discard remaining unsorted oocytes in the sink and throw away the other dishes

11. Place good oocytes in 17°C incubator