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Adult zebrafish were maintained at
28.5°C 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 28.5°C.
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 28.5°C. 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 40.0°C water
bath and embryos were heat shocked for 30 minutes. After
this period, the embryos were returned to medium at 28.5°C
and incubated for 24 hours.
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.
Last Modified: 10 May 2004
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