limb bud culture

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Detailed Laboratory Procedures²Week 1: Preparation and culture of the limb bud.

1. In 6 well plates, place tissue culture filter inserts over approximately 1.8ml of culture media.

2. Prepare the 6-day donor egg by sterilizing the outside with 70% ethanol. Carefully remove the embryo to sterile Howard Ringer's solution and remove membranes.

3. Record the stage of development.

4. Pinch off each limb bud and transfer to a culture dish using siliconized pasteur pipets. Place buds on top of the filter inserts. Remove any media on top of the filters.

5. Place cultures in an incubator at 37ºC with 5% CO₂. Observe growth periodically over the next week.

Week 2: Staining for limb cartilage.

1. Fix limbs in 5% TCA for 1 hour at room temperature.

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. Wash with 85% ethanol, 95% ethanol, 100% ethanol.

6. Clear by washing 1-3 times with methyl salicylate.

7. Observe cartilage and compare to control limbs that developed in vivo.

@Cebra-Thomas, 2000

Last Modified: 2 August 2001

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		Detailed Laboratory Procedures²Week 1: Preparation and culture of the limb bud.
		1. In 6 well plates, place tissue culture filter inserts over approximately 1.8ml of culture media. 2. Prepare the 6-day donor egg by sterilizing the outside with 70% ethanol. Carefully remove the embryo to sterile Howard Ringer's solution and remove membranes. 3. Record the stage of development. 4. Pinch off each limb bud and transfer to a culture dish using siliconized pasteur pipets. Place buds on top of the filter inserts. Remove any media on top of the filters. 5. Place cultures in an incubator at 37ºC with 5% CO₂. Observe growth periodically over the next week.
		Week 2: Staining for limb cartilage.
		1. Fix limbs in 5% TCA for 1 hour at room temperature. 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. Wash with 85% ethanol, 95% ethanol, 100% ethanol. 6. Clear by washing 1-3 times with methyl salicylate. 7. Observe cartilage and compare to control limbs that developed in vivo.