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Routine Transmission Electron Microscopy (TEM) Staining Protocol for Cultured Cells
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Routine Transmission Electron Microscopy (TEM) Staining Protocol for Cultured Cells


  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.


  1. 50% ethanol  15min
  2. 70% Ethanol  15min
  3. 95% Ethanol  15min
  4. 100% Ethanol  2x15min
  5. 100% Propylene oxide  2x15min
  6. 1:1 EMBed 812 and Propylene Oxide for1-2 hour.
  7. 2:1 EMBed 812:Propylene Oxide overnight in dessicator with top off.


Embed in Beam capsules, and bake in 60 ºC oven for 48 hours.


Semithin (thick section, 0.5-1 um) and toluidine blue staining. Observe the thick section, and trim EM block further if needed. Then ultrathin sectioning, and collected on grids.


Staining grids with uranyl acetate (see reagent below) for 15 minutes, rinse with distilled water and then staining with lead citrate (see reagent below) for 3-5 minutes, rinse with distilled water.

Solutions and Reagents:

0.2M PB, pH 7.4:

Na2HPO4 ------------------------ 21.8 g

NaH2PO4 ------------------------ 6.4 g

Distilled water ----------------- 1000 ml

4F1G Fixative (4% Formaldehyde & 1% Glutaraldehyde in 0.1 M PB, pH 7.4):

0.1M PB, pH 7.4 --------------- 88 ml

37-40% Formaldehyde ----- 10 ml

50% Glutaraldehyde --------- 2 ml  

8% (0.2M) Sucrose in 0.1M PB:

Sucrose ------------------------- 8 g

0.1 M PB ------------------------ 100 ml

1% Osmium in 0.1M PB:

2% Osmium -------------------- 5 ml

0.2M PB, pH7.4 --------------- 5 ml

5% Uranyl Acetate Solution:

To prepare 50 ml, add 2.5 g of uranyl acetate to 50 ml of distilled water. Cover with foil and stir overnight. Add 10 drops of glacial acetic acid. Store solution in 4 ºC.

Reynold’s Lead Citrate Solution:

To prepare 50 ml, add chemicals in distilled water in following order

Lead nitrate ------------------------------ 1.33 g

Sodium citrate, dihydrate -------------- 1.76 g (solution becomes cloudy when sodium citrate is added)

1N NaOH ----------------------------------- 5 ml  (solution becomes clear when NaOH is added)

Distilled water ----------------------------- 30 ml

Stir for 10 minutes to dissolve and add additional 15 ml of distilled water. Store solution for 3-6 months at 4 ºC. Note: the mount of NaOH is very important. Too much will make solution cloudy.

EMBed 812:

Final VolumeEMBed-812DDSANMADMP-30 (2%)
20.91 ml10 ml4.5 ml6 ml0.41 ml
31.37 ml15 ml6.75 ml9 ml0.62 ml
41.82 ml20 ml9 ml12 ml0.82 ml
52.28 ml25 ml11.25 ml15 ml1.03 ml

        Mix all four components in plastic beaker and stir with wood stick.

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