Whole mount antibody staining ofzebrafish embryos formarkers ofsegmentation
1. Dechorionate 26 hr embryos (pharyngula stage) carefully with two fine forceps.
Transfer to fixative (1% formaldehyde in PBS). Fix for 1 hour rocking at 4oC.
2. Wash with 5 ml 0.1% BSA in PBS for 10 minutes. Wash 3X with 5 ml PBS,
10 minutes each.
3. Incubate overnight at 4oC (cold room) in 0.5 ml primary antibody in 0.2% saponin in PBS.
Primary antibodies: (A) znp-1 @ 1/2000 (primary motoneurons)
(B)F6@ 1/500 (somite boundaries)
(C) no antibody (Control)
4. Wash for a minimum of 2 hours with several changes of 0.2% saponin in PBS (can store in cold room at this point).
5. Incubate overnight with 0.5 ml secondary antibodyconjugated to peroxidase (HRP) (Goat anti- mouse IgG+IgM-HRP) @ 1/500at 4oC.
6. Wash for a minimum of 2 hours with several changes of 0.2% saponin in PBS (can store in cold room at this point).
7. Wash with 1 ml HRP substrate buffer.
8. Develop with HRP substrate (metal-enhanced DAB). Monitor under dissecting microscope after 5 minutes. When brown color is clearly visible, stop reaction by washing with PBS.
9. Clear embryo by soaking in 1:1 glycerol:CMFET, followed by 1:4 glycerol:CMFET.
Observe with dissecting microscope, and mount with depression slides to observe with compound microscope.
