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Immunofluorescent staining of archenteron cells
(all samples)

1. Allow 2 tubes of embryos to settle for 10 minutes.
Gently remove most of the liquid.
2. Add 200 ml 5C7 antibody (stains vegetal plate and archenteron) to one tube and 200 ml 10%
normal goat serum to the other. Let sit for 45 minutes at room temperature. Embryos will
settle.
3. Remove most of liquid. Add 1 ml ASW to wash. Allow to settle, remove liquid. 4. Add 200 ml
Fluorescein-congugated Goat anti-mouse IgG to both tubes. Let sit for 45 minutes at room
temperature. Embryos will settle.
5. Remove most of liquid. Add 1 ml ASW to wash., Allow to settle, remove liquid. Add 100 ml
PBS.
6. Transfer 10 ml of each sample to microscope slides. Check that there are embryos.
Coverslip. Examine using epifluorescence.
7. Observe differences from control.


Results

Throughout the course of our experiment, we observed our embryos at five different times. The first observation was made only 30 minutes after fertilization. The only sample that was not observed at this time was sample #4 because it was only irradiated 30 minutes after fertilization. At this 30 minute mark, we saw normal development in the control. Cells were already beginning to undergo the first cleavage. The other two irradiated samples had not yet begun to cleave at this point.

After 5 hours, the control sample showed normal development at the blastula stage. Sample 2 exhibited a huge variance in stage. Some were halted after first cleavage, while others had divided several times. Most cells that did divide, had done so unevenly. Sample 3 exhibited many unusually small cleavages. Sample 4 contained cells that appeared to have divided anywhere from two to six times. The divisions in this sample were extremely irregular.

© 2000 Cebra-Thomas

Last Modified: 25 April, 2000


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