Glutaraldehyde Fixation and SEM Preparation of Cells

From LPDwiki

About this protocol: This protocol outlines the steps and materials involved in preparing cells to be imaged under the scanning electron microscope.

Necessary Materials

1. 100% Ethanol

  • Purchased from Vet Med Stockroom (in Vet Med 3B)

2. 25% Glutaraldehyde in Water

  • Purchased Sigma

3. Hexamethyldisilazane (HMDS)

4. Deionized Water

5. Phosphate Buffered Saline Solution

6. Gold Sputter (SEM grade)

Procedure

1. Wash cells with warm PBS three times.

2. Fix in 2.5% glutaraldehyde in warm PBS for 20 min.

3. Wash with PBS three times.

4. Wash with DI water.

5. Soak for 5 min at RT consecutively in absolute ethanol (20%, 50%, 75%, 90%, 95%, 100%) in DI water.

6. Soak in 1-1 (HMDS-ethanol) 5 min and 100% HMDS for 10 minutes (If not ready to dry, store in absolute ethanol in fridge sealed with parafilm).

7. Remove some of the HMDS and let it evaporate for it to dry.

8. Coat with very thin gold (~a few nm or so) and image.

Cleaning Up