Notes

1. Dissecting instruments need to be kept clean to guard against infection. Wipe instruments before use and before putting away with 70% alcohol. Another useful tip that can cut down on infection, as well as preventing the embryo from drying under the bright lights used to illuminate it, is to add a small drop (up to 10 ||L) of medium containing antibiotics to the embryo during or after the operation.

2. For separation of mesenchyme and ectoderm, it is very important that all solutions and the tissue be kept very cold. Otherwise the tissue can become sticky. In early days of tissue disaggregation, the sticky gel associated with cells following trypsin treatment was thought to be glue that held cells together—later this glue was shown to be digested by DNase! Batches of trypsin may vary so it is wise to test out new batches in a trial run.

3. Until one gets used to looking at the embryo in the egg, it can be difficult to make out the apical ridge and to find tissue/cells that have been transferred into the egg. One way of increasing contrast is to use a green filter in the light path, but this reduces the light intensity. It is also possible to use a very weak solution of a vital dye, such as nile blue sulfate. Add a few drops of 0.1-0.2% nile blue sulfate in PBS to the embryo in the egg, or place tissue in a similar solution for a few seconds so that it is stained a very light blue. Be aware that nile blue sulfate at high concentrations can be toxic. We have found that if polarizing regions are stained, their signaling ability is reduced in parallel with the deepness of the shade of blue.

4. For polarizing region grafts to work most effectively, they must be placed in contact with the apical ridge. It is therefore important that the apical ridge is separated as cleanly as possible from the underlying mesenchyme. If the wing bud bleeds while you are lifting the ridge, you are cutting too far away from the apical ridge and into the marginal sinus that runs about 100 |im away from the chick wing ectoderm (33).

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