1 2 3 4 5 6 7

Methods

Fish care

Adult zebrafish were maintained at 28.5C on a 14h light/10h dark cycle. Embryos were collected from natural spawnings and maintained in embryo medium (NaH2PO4 0.01M 10mL; Na2HPO4 0.01M 10mL; Sodium citrate 0.1M 20mL; CaCl2 0.1M 15mL; dH20 945 mL) at 28.5C.

 

Treatment

Embryos were transferred into test medium using wide-bore pasture pipettes. Embryos were grouped by stage as observed through a light microscope and allowed to develop at 28.5C. After reaching somite stage, approximately 10-11 hours after fertilization, embryos were removed and placed in glass test tubes containing embryo medium. The test tubes were then placed in a 40.0C water bath and embryos were heat shocked for 30 minutes. After this period, the embryos were returned to medium at 28.5C and incubated for 24 hours.

 

Visualization

Embryos were dechorionated using foreceps and a light microscope. To visualize motoneuron axons, embryos were fixed in 4% paraformaldehyde (PFA) overnight, followed by washing in PBS. They were then stained with znp-1 antibody, F6 antibody, goat anti-mouse secondary antibody conjugated to HRP, and stored in glycerol:CMFET according to methods detailed in The Zebrafish Book (Westerfield, 1993).

Embryos were mounted on depression slides for observaiton under a microscope and photographs were taken using the Olympus 1000.

©Cebra-Thomas, 2000

Last Modified: 10 May 2004

[Lab Protocols | Students | Cebra-Thomas | Course | Links ]