Notes

1. Micropipet tips easily become blocked when the DiI solution comes into contact with aqueous solutions and/or is covered in cellular debris. The easiest way to unblock the pipet is to stroke the pipet tip very gently with a small wisp of tissue paper soaked in alcohol. Do this under a dissecting microscope. When the pipet is unblocked, a small deposit of DiI will be drawn out onto the tissue paper.

2. Minimize the time the pipet tip spends in the embryo. The longer it is in contact with embryo or aqueous solution, the more likely it is to block up.

3. If the targeted tissue has sufficient depth, then a small withdrawal of the pipet tip after its initial insertion into the tissue will often facilitate deposition of the dye.

4. If your pipet does not readily push through superficial cell layers to reach deeper cells, then you can first make a small superficial hole with a sharpened tungsten needle and follow this with your DiI pipet.

5. If a small blob of Dil forms on the tip of the electrode as you are trying to inject, first disconnect the battery to stop current flow and wait a short while (30 s or so). The DiI blob may simply dissolve in to the surrounding cells. If it does not dissolve, it will probably stick to the electrode as it is withdrawn from the tissue. Do not worry. You will still have labeled plenty of cells. Use a fresh electrode for the next injection.

6. Your electrode should only ever touch the cells you want to label. Try not to have to reposition it. Its amazing how easily you can inadvertently label cells simply by touching them with the electrode even when the battery is disconnected. This is especially true if the electrode is not a new one.

7. Use a new electrode at least every three embryos. The best electrode is a new one.

8. Poor electrode design is the most likely reason for lack of success.

9. Vibration must be eliminated. Make sure microscope and manipulator are solidly attached to the base plate and that the manipulator only moves when you want it to move, not for instance, when you grab the manipulator, but have yet to turn any knobs.

10. Watch out for embryos that float very slowly across the field of view. This means they are not perfectly balanced within the egg and the egg should be slightly rotated in an appropriate direction to eliminate this drift. You cannot microinject a moving target (well, the author cannot anyway).

Was this article helpful?

0 0

Post a comment