FITC-tyramide synthesis (provided by L.Davidson)

The plan: To link the FITC 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:

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:

1. Make a 10 mg/ml stock of your NHS ester in DMF
40 mg FITC NHS ester (MW = 473)
4 ml DMF

2. Make DMF-TEA solution
1 ml DMF (dimethylformamide)
10 ul TEA (triethylamine)

3. Make Tyramide solution
10 mg tyramide
1 ml DMF-TEA

The Reaction:

4 ml FITC 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