Developmental Biology- Heatshock causes disruption in somite development

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Methods

Fish care

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.

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 28.5°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.

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

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		Methods
		Fish care 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. 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 28.5°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. 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.