Flourescin Tyramide Synthesis: Difference between revisions
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''' | '''Provided by: L. Davidson''' | ||
The plan: To link the fluorescein fluor onto tyramide. The reactive esters are effective couplers if they are in good condition. The reaction works best in an anhydrous environment - use fresh solvents. | '''The plan:''' | ||
To link the fluorescein fluor onto tyramide. The reactive esters are effective couplers if they are in good condition. The reaction works best in an anhydrous environment - use fresh solvents. | |||
The materials: | '''The materials:''' | ||
fluorescein-NHS ester Pierce 46100 40 mg | fluorescein-NHS ester Pierce 46100 40 mg | ||
tyramide Sigma T-2879 1 gm | tyramide Sigma T-2879 1 gm | ||
| Line 11: | Line 12: | ||
Ester stock Caution: NHS esters are very unstable. Once opened dissolve entire contents in DMF and use at once. If you cannot use all of the solution keep tightly sealed and store frozen in the dark - protect from absorbing water. | Ester stock Caution: NHS esters are very unstable. Once opened dissolve entire contents in DMF and use at once. If you cannot use all of the solution keep tightly sealed and store frozen in the dark - protect from absorbing water. | ||
'''The Solutions:''' | |||
# Make a 10 mg/ml stock of your NHS ester in DMF (40 mg fluorescein NHS ester (MW = 473), 4 ml DMF) | |||
The Solutions: | # Make DMF-TEA solution (1 ml DMF (dimethylformamide), 10 ul TEA (triethylamine)) | ||
# Make Tyramide solution (10 mg tyramide, 1 ml DMF-TEA) | |||
# Make a 10 mg/ml stock of your NHS ester in DMF | |||
40 mg fluorescein NHS ester (MW = 473) | |||
4 ml DMF | |||
# Make DMF-TEA solution | |||
1 ml DMF (dimethylformamide) | |||
10 ul TEA (triethylamine) | |||
# Make Tyramide solution | |||
10 mg tyramide | |||
1 ml DMF-TEA | |||
'''The Reaction:''' | |||
4 ml fluorescein NHS in DMF | 4 ml fluorescein NHS in DMF | ||
1.37 ml tyramide solution | 1.37 ml tyramide solution | ||
| Line 38: | Line 25: | ||
This is the best reagent to date, and also vastly cheaper. As Lance put it, one batch will make enough tyramide to "wholemount a cow". Diluted 1:100 = 1 ul per reaction, 10 ml = 10,000 reactions. | This is the best reagent to date, and also vastly cheaper. As Lance put it, one batch will make enough tyramide to "wholemount a cow". Diluted 1:100 = 1 ul per reaction, 10 ml = 10,000 reactions. | ||
Papers and other sites: | '''Papers and other sites:''' | ||
*Davidson, L. A., and Keller, R. E. (1999). Neural tube closure in Xenopus laevis involves medial migration, directed protrusive activity, cell intercalation, and convergent extension. [http://www.ncbi.nlm.nih.gov/pubmed/10498689?dopt=Abstract| Development, 126:4547-4556] | |||
*Zhou, X. and Vize, P.D. (2004). Proximo-distal specialization of epithelial transport processes within the Xenopus pronephric tubules. [http://www.ncbi.nlm.nih.gov/pubmed/15223337?dopt=Abstract| Developmental Biology 271: 322-338] | |||
*Davidson, Keller, and DeSimone (2004) Patterning and tissue movements in a novel explant preparation of the marginal zone of Xenopus laevis. [http://www.ncbi.nlm.nih.gov/pubmed/15183313?dopt=Abstract| Gene Expression Patterns Jul;4(4):457-66] | |||
Davidson | *[http://www.ncbi.nlm.nih.gov/pubmed/15183313?dopt=Abstract| Lance Davidson's flourescent in situ methods page] | ||
*Vize, P.D., McCoy, K.E., and Zhou, X. (2009). Multichannel wholemount fluorescent and fluorescent/chromogenic in situ hybridization in Xenopus embryos. [http://www.ncbi.nlm.nih.gov/pubmed/19498377| Nat Protoc. 4(6): 975-983.] | |||
Latest revision as of 11:14, 12 June 2012
Provided by: L. Davidson
The plan: To link the fluorescein fluor onto tyramide. The reactive esters are effective couplers if they are in good condition. The reaction works best in an anhydrous environment - use fresh solvents.
The materials: fluorescein-NHS ester Pierce 46100 40 mg tyramide Sigma T-2879 1 gm dimethyl formamide Sigma T-8654 triethylamine Sigma T-0886
Ester stock Caution: NHS esters are very unstable. Once opened dissolve entire contents in DMF and use at once. If you cannot use all of the solution keep tightly sealed and store frozen in the dark - protect from absorbing water.
The Solutions:
- Make a 10 mg/ml stock of your NHS ester in DMF (40 mg fluorescein NHS ester (MW = 473), 4 ml DMF)
- Make DMF-TEA solution (1 ml DMF (dimethylformamide), 10 ul TEA (triethylamine))
- Make Tyramide solution (10 mg tyramide, 1 ml DMF-TEA)
The Reaction: 4 ml fluorescein NHS in DMF 1.37 ml tyramide solution
mix and incubate in dark at room temp for 2 hours add 4.6 ml ethanol, store in dark at 4 or -20
This is the best reagent to date, and also vastly cheaper. As Lance put it, one batch will make enough tyramide to "wholemount a cow". Diluted 1:100 = 1 ul per reaction, 10 ml = 10,000 reactions.
Papers and other sites:
- Davidson, L. A., and Keller, R. E. (1999). Neural tube closure in Xenopus laevis involves medial migration, directed protrusive activity, cell intercalation, and convergent extension. Development, 126:4547-4556
- Zhou, X. and Vize, P.D. (2004). Proximo-distal specialization of epithelial transport processes within the Xenopus pronephric tubules. Developmental Biology 271: 322-338
- Davidson, Keller, and DeSimone (2004) Patterning and tissue movements in a novel explant preparation of the marginal zone of Xenopus laevis. Gene Expression Patterns Jul;4(4):457-66
- Vize, P.D., McCoy, K.E., and Zhou, X. (2009). Multichannel wholemount fluorescent and fluorescent/chromogenic in situ hybridization in Xenopus embryos. Nat Protoc. 4(6): 975-983.