3/09/03

Enrobement of dispersed cells for TEM

A method for aggregating dispersed cells such as RBCs for TEM processing.

Prepare stock solutions

15% BSA in 0.1M cacodylate buffer (150 mg/ml)

5% glutaraldeyde in 0.1M cacodylate buffer (200 µl of 25%/ml)

Fixation

Sediment cells in eppendorf tube at low speed. Decant most of supernatant and resuspend cells in fixative.

Enrobement

1. Sediment fixed cells and decant.
2. Resuspend cells in 0.5 ml of 15% BSA solution at 4°C.
3. Add 0.5 ml of 5% glutaraldehyde, mix gently, and sediment cells immediately at 10°C.
4. Incubate for 15 to 30 min at room temperature (until gel formation)
5. Cut out pellet, remove empty BSA, mince and wash segments in buffer.