3 4 5
-Fertilized chicken eggs
were incubated in the laboratory at 37o C with
constant humidity until the desired stage (approximately 20
preparation and administration:
- 5 uL of Cyclopamine in
2-hydroxypropylcyclodextrin (HPCD) was administered to the
embryo by injecting it through the vitelline membrane with a
small pipette, through a small hole carefully cut in the
- A solution of 45 % HPCD in
PBS wasadministered in a
similar fashion to all the control embryos.
- Embryos were allowed to
continue developing in the incubator at 37 oC for
an additional 48 hours.
-Embryos were collected
at 68 hours after initial incubation, or 48 hours after
treatment. - Embryos were then washed with Howard's
Ringer solution in a petri
dish. Any extra- embryonic membranes were
carefullyremoved with fine
- Embryos were covered with
an appropriate volume of PI working solution (10 mg/ml PI in
PBS) for approximately 10 minutes.
- Embryos were washed with
Ringer and mounted on a
depression slide. - Embryos were observed under a
fluorescent microscope for the presence of dead cells in the
tail bud and craniofacial regions.
-The embryos that were
allowed to develop for a total of five days with cyclopamine
were not stained and later observed under a dissecting
microscope and photographed.
Last Modified: 3 May 2001