Objective Intro Materials Procedure Results Discussion Lit. Cited Prep Sheet
		Embryo Harvesting 1) Induce adult zebrafish to mate at the beginning of their light cycle by covering the bottom of the aquarium with a layer of marbles the night before. This will both excite the fish and protect the embryos from predation. Leave overnight. 2) Harvest the embryos between one and three hours after the fish have awakened (at the appearance of light) by using a suction vacuum to clean the bottom, removing embryos from under the marbles into a filter. 3) Wash embryos from filter into a dish of aquarium water. Sort the embryos from extraneous debris by using a wide-mouth pipet to remove them into a Petri dish of Zebrafish Embryo Medium (ZE) solution. 4) Stage the embryos and allow to develop to desired stage. Treatment 1) Prepare solutions of 0.05M valproic acid (VA) in ZE and 0.1M VA in ZE. Use ZE without VA as a control. 2) Pour solutions into six separately labeled dishes. There will be two dishes of each solution (three destined for F6 staining and three destined for alcian green staining) 3) At seven hours after fertilization (the time of first somitogenesis), add between ten and twenty embryos to each of the six dishes. 4) Incubate embryos at 28°C. 5) At 33 hours after fertilization, fix (follow the F6 staining protocol below) one dish of embryos exposed to each concentration of VA and one control dish. Follow the protocol for F6 antibody staining and photograph the stained embryos. F6 stains for somite boundaries. 6) At four days after fertilization, fix (follow the Alcian Green staining protocol) one dish of embryos exposed to each concentration of VA and one control dish. Alcian binds to cartilage. Photograph stained embryos.   F6 whole mount antibody staining for Zebrafish 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 4° C. 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 4° C (cold room) in 0.5 ml primary antibody in 0.2% saponin in PBS. Use F6@ 1/500 (somite boundaries). 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 antibody conjugated to peroxidase (HRP) (Goat anti- mouse IgG + IgM-HRP) @ 1/500 at 4° C. 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 diamino benzidine with NiCl [Pierce Chemical]). 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 4:1 glycerol:CMFET. Observe with dissecting microscope, and mount with depression slides to observe with compound microscope. back to treatment Alcian green stain for cartilage in Zebrafish Alcian green stain binds to cartilage, but not calcified bone. This allows study of early skeletal formation. 1) Fix embryos in 5% TCA for 1 hr. at room temp. 2) Wash 3 times with PBS. 3) Immerse in Alcian green stain for 4 hours with gentle agitation. 4) Wash 3 times with 70% ethanol. 5) Clear embryos by washing first in 1:1 glycerol:CMFET, then in 4:1 glycerol:CMFET. 6) Photograph embryos ------>Alcian green stain: 0.1g Alcian green (100 ml)70 ml100% ethanol 1 ml conc. HCl 29 ml dH2O back to treatment © Cebra-Thomas, 2001 Last Modified: 14 July 2014 [Lab Protocols | Students | Cebra-Thomas | Course | Links ]