MsoDockBottom

Bio 33 Embryology
Laboratory 4-5: Zebrafish early development and segmentation

Zebrafish are teleost fish that develop by meroblastic cleavage on top of a large yolk mass. Zebrafish eggs are fertilized as they are laid, usually at "dawn" (10:30 am for our fish). In this laboratory we will observe early cleavage and blastula stages (and gastrulation if you come back in the evening). We will also fix post-gastrulaton embryos, and use antibodies to examine segmentation of the trunk somites and its effect on primary spinal motoneurons. We will start the staining in the first week, and develop the color reaction in the second week.

Whole mount antibody staining
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.


		Bio 33 Embryology Laboratory 4-5: Zebrafish early development and segmentation Zebrafish are teleost fish that develop by meroblastic cleavage on top of a large yolk mass. Zebrafish eggs are fertilized as they are laid, usually at "dawn" (10:30 am for our fish). In this laboratory we will observe early cleavage and blastula stages (and gastrulation if you come back in the evening). We will also fix post-gastrulaton embryos, and use antibodies to examine segmentation of the trunk somites and its effect on primary spinal motoneurons. We will start the staining in the first week, and develop the color reaction in the second week. Whole mount antibody staining 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.