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Procedure:

1. Dechorionate 24 hour zebrafish embryos in the pharyngula stage with 2 fine forceps.

2. Fix approximately of the embryos with 4%, and the other half in 1% paraformaldehyde in PBS for one hour.

3. Wash embryos 3x in 5 ml PBS to remove fixative

4. Incubate embryos in PBS with goat serum and 0.2% saponin

5a. Add the primary antibody,ZNP-1 at 1/2000 to half the cells with 4% paraformaldehyde,keep the other half as a control,incubate for 24 hours.

5b. Add the primary antibody,F6 at 1/500 to half the cells with 1% paraformaldehyde,keep the other half as a control,incubate for 24 hours.

6. Wash embryos with several changes of 5 ml PBS to remove antibody

7. Incubate with the secondary antibody,Fluorescent Goat anti-mouse IgG+ for 45 minutes

8. Wash with several changes of PBS

9. Mount with depression slides and observe stained embryos using the fluorescent microscope.

10. Look for individual brightly stained cells in the tail region.

 

Figure 1.

Dechorionated Zebrafish embryo at approximately 24 hours

 

 

 

@ Cebra-Thomas, 2001

Last Modified: 2 August 2001


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