Procedure
Procedure
1. Obtain several Hamburger & Hamilton stage 24 (d/d) axolotl embryos that have undergone gastrulation.
2. Remove jelly membrane using sharpened forceps.
3. Transfer dejellied embryos to 3 mL Holtfreter's solution.
4. Divide embryos into four experimental group Petri dishes and one control group Petri dish. Each dish should have a 3 mL Holtfreter's solution substrate. Label the dishes "control," "50 uM cyclopamine," "100 uM cyclopamine," "30 uL ethanol," and "60 uL ethanol."
5. Use cyclopamine at 2 mg/mL in ethanol as a stock. Dilute this solution in 10% HBSt to create two final solutions with concentrations 50 uM and 100 uM.
6. Add the 50 uM cyclopamine solution to the "50 uM cyclopamine" group, and add the 100 uM solution to the "100 uM cyclopamine" group.
7. Add 30 uL of ethanol to the "30 uL ethanol" group and add 60 of ethanol to the "60 uL ethanol" group.
8. Retain the control embryos in 3 mL Holtfreter's solution.
9. Keep embryos at 25° C for 24 hours.
10. Take still pictures and make observations.
11. Continue to make observations and take pictures every 24 hours until four observations total have been made.