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TEM PREP for Tissue Culture Cells

(monocytes and dendritic cells)
Neil Fanger, Microbiology/Borwell)

Spin Cells/pellet 500-1000rpm on clinical centrifuge, maximum, for each step.

MAKE SURE CELLS ARE IN POLYPROPYLENE TUBES, NOT POLYSTYRENE

1. Clinical centrifuge cells to concentrate. Fix in 2% GTA in 0.1M NaCac with 0.1 M Sucrose, pH 7.4 (buffer osmolarity is 300 and total is 510 mosmols) for 15 minutes. Resuspend and add fresh 2% GTA in 0.1M NaCac with 0.1 M Sucrose, pH 7.4. Continue fixing for 2 hours RT. Or, if necessary, store in this fixative at 4 C until ready to prep.

2. Rinse in 0.1M NaCac pH 7.4; 3X 5 mins each.

3. Postfix in aqueous 1% OsO4 for 1hr. at RT.

4. Rinse in dH20.

5. En-block stain with 2%UAaq for 1 hr. RT, in the dark.

6. Dehydrate thru ETOH series: 30%, 50%, 70%, 95%, 5 mins. ea.

7. Three 15 min. changes in 100% ETOH.

8. Two 10 min. changed in Propylene oxide(PO).

9. Cells are immersed in LX112:PO - 1.5:1 {10.2 ml:6.8 ml}. Cap for 2 hrs. Remove caps and place in vacuum desiccator overnight.

10. Transfer cells to Beem capsules and fill with fresh LX112. Spin down for 30 minutes at 1,000 RPM to get cells to botto of capsule. Place in vacuum desiccator overnight

11. Polymerize at 60 C for 48 hrs. Remove blocks from heat to cool.

12. Stain thin sections with 2% Uranyl Acetate(UA) in MEOH for 20 minutes and Reynold's Lead Citrate for 5 minute.

Last Updated: 10/2/08