Routine TEM Staining Protocol for Cells
Fixation: 1. Fix cell suspension or free cells in 4% formaldehyde and 1% glutaraldehyde in 0.1 M PB (pH 7.4) by mixing equal volume of fixative and cell suspension. 2. Transfer cells to centrifuge tube and spin for 10 minutes. A nice, tight pellet will be formed (Re-spin if necessary during processing). Carefully pipette off fixative. Add fresh fixative for at least 2 hours or overnight. 3. Pipette off fixative. Replace with 8% (0.2M) sucrose in 0.1 M PB 3x15 minutes or overnight at 4 C (Samples can be kept in sucrose for a long time). 4. Post-fix with 1% OsO4 in 0.1 M PB for 1 hour. 5. Pipette off OsO4, Rinse in 0.1 M PB 3x10 minutes. Dehydration: 1. 50% ethanol 15min 2. 70% Ethanol 15min 3. 95% Ethanol 15min 4. 100% Ethanol 2x15min 5. 100% Propylene oxide 2x15min 6. 1:1 Epon and Propylene Oxide for1-2 hour. 7. 2:1 Epon:Propylene Oxide overnight in dessicator with top off. Embedding: 8. Embed in Beam capsules. 9. 60 C oven for 48 hours. |