Flourescin Tyramide Synthesis and Wnt-2: Difference between pages

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'''{rovided by: L. Davidson'''
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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
 
Lance Davidson's flourescent in situ methods page

Latest revision as of 06:00, 10 February 2010

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