Purpose#

Technique used when one needs to patch a cell body that is typically occluded by a tightly-associated glial sheath. This type of sheath cannot be removed by traditional de-sheathing or cleaning techniques, and is more common in cells that are more superficial in the brain. You can identify whether your cell is sheathed based on the appearance (cell body appears textured rather than smooth, with small dark puncta on the surface) and whether it is even possible to break in to the cell even with a good seal and large pipette tip.

Here is an example of a heavily sheathed cell body

Preparation#

1. Dispase is located in the -20C freezer as 20uL aliquots at 20mg/mL (in 1XPBS)
2. Dilute an aliquot to 1mg/mL with fresh fly saline
3. Filter the solution into a fresh tube
4. Store at 4C for up to ~2weeks

Application#

1. With a large cleaning pipette, clean around your cell until it is exposed
2. With a small to medium sized cleaning pipette, hose down your cell with dispase for ~10-20 seconds (depends on how sheathed your cell is)
3. If application was too short, the sheath around your cell won’t clear
4. If application was just right, the sheath will clear on at least part of your cell and the cell surface should look clean
5. If application was too long, the sheath will clear but your cell will look digested and shrunken
6. To remove your cell’s sheath, either:
7. Use a large cleaning pipette to blow away the broken up sheath
8. Use a small cleaning pipette to pull away the broken up sheath 1. If you use the latter technique, use a large pipette after to clear the cell surface for patching