1 2 3 4 5 6 7

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Procedure

Foil technique

1. Prepare microtools for surgery.

2. Prepare 1% agarose-coated operating dish; rinse with 100% HBSt and antibiotics.

3. Autoclave a small piece of aluminum foil; this foil will be used to bisect the morphogenetic fields of the heart.

4. Sort embryos by age, stop development of stage 15 axolotl embryo by placing in 4°C. The developing embryo is sensitive to temperature and will develop faster at higher temperatures (Hamburger, 1960).

5. Manually dejelly embryo in Steinbergs solution as demonstrated in video on next page. Use fine forceps to break hole in the hard jelly coat. Maneuver embryo out. Be careful not to puncture the embryo!

6. Transfer embryos into well of operating dish containing increasing concentrations of HBSt and antibiotics. Remove half the solution with pipetteman and fill with 100% HBSt. After a minute remove half the solution and fill with 100% HBSt. Allow embryo to sit 2 minutes in high salt solution. This will cause the vitelline membrane to swell and make it easier to see and remove as shown in the video. Demembranate embryo in 100% HBSt by carefully grabbing near neural crest cells with both tweezers and pulling to form a hole as described in the dejellying step above.

7. Dip all tools into 70% EtOH and then into sterile HBSt before using.

8. Hold recipient embryo ventral side up with hair loop and pick a small slit down the midline of the embryo along the anterior-posterior axis with eyebrow microscalpel or tungsten microscalpel.

CAUTION: Be gentle. Do not slice through the entire embryo and be careful not to cause inner cells to spew out of embryo!

9. Place foil in the incision deeply enough so that it does not fall out but not so deeply that it cuts through the embryo. Observe development until heart forms.

10. Leave one embryo with only the incision but no foil, and one embryo uncut as controls.

11. While the high Ca2+ concentration of the HBSt enhances healing by saturating the Ca2+ binding sites of cadherin molecules, a high salt concentration also causes developmental abnormalities. Therefore, after embryos heal from surgery, gently replace 100% HBSt with 50%, then 20% HBSt. Change solution concentrations after 12 hrs.

12. Collect pictures and movies daily using a microscope equipped with a camera and recording cameras.

© Cebra-Thomas 2004

Last Modified: May 13 2004


[Lab Protocols | Students | Cebra-Thomas | Course | Links ]