TEM Immunogold Labeling Protocol - Post-embedding Method Using L.R. White Embedding Medium






1.   Fixation: fix specimen with 4% formaldehyde in 0.1M PB, pH 7.4 for 12-24 hours. For free cells or cell suspension, fix cell pellet for 2-4 hours.

2.   Rinse in 0.1M PB for 3x10 min.

3.   Quench Free Aldehyde: incubate specimen in 0.1M Glycine Solution for 20 minutes to quench the   free aldehyde groups. Note: this step may not be necessary.

4.   Incubate in 0.2M Sucrose Solution for 2x15min or overnight at 4C. Note: specimen can be stored in the sucrose solution for several weeks.

5.   Rinse in 0.1M PB briefly.

6.   Dehydration: incubate specimen in two changes of 70% ethanol 30 minutes each. Then incubate in L.R. White and 70% ethanol (2:1) mixture: slowly add one part of 70% ethanol drop by drop to two parts of L.R. White, and shake gently to avoid mixture becomes milky. Incubate specimen in the mixture for 1 hour.

7.   Transfer specimen in three changes of pure L.R. White for 1 hour each. Keep specimen in the last change of L.R. White overnight at room temperature (specimen can be stored in L.R. White at 4 C for weeks if necessary).

8.   L.R. White Embedding: place specimen in bottom of Gelatin Capsule, fill up with L.R. White to the brim, and slide the other half of the capsule on. Polymerize in 60 C oven for 24-48 hours or longer.

9.   Trimming: trim EM blocks and cut 0.5um thick sections and stain with toluidine blue if necessary, and then select the region of interest and trim blocks further to the appropriate size.

10. Sectioning: cut ultrathin sections at 60-90 nm.

11. Collect sections on formvar-carbon coated nickel grids (5 grids per specimen), and allow the grids to dry overnight prior to staining.

12. Pretreatment: steam grids in Epitope Unmasking Solution (based on LM test or use citrate buffer pH 6.0) for 20 minutes, and allow grids to cool for 20 minutes. This step may not be necessary.

13. Rinse sections for 2x2 min by place grids on large droplets of TBS-Tween.

14. Serum Blocking: incubate sections in IHC Ultra Serum Block – Species match secondary antibody for 30 minutes.

15. Primary Antibody: incubate sections on droplets of primary antibody diluted in Universal Antibody Diluent for 2 hours at room temperature. Note: primary antibody dilution is usually 10 times more concentrated than immunostaining at LM level.

16. Rinse sections on large droplets of TBS-Tween for 6x2 min.

17. Secondary Antibody: incubate sections on droplets of biotinylated secondary antibody (1:50, Vector Lab) in Universal Antibody Diluent for 1 hour at room temperature.

18. Rinse on large droplets of TBS-Tween for 6x2 min.

19. Streptavidin-Gold: incubate sections with gold conjugated streptavidin (1:20, EMS) in TBS, pH 7.6 for 1 hour. Note: do not use BSA or serum containing solution/reagent to dilute streptavidin-gold since BSA or serum may contain biotin, therefore reduce streptavidin-gold affinity to biotinylated secondary antibody.

20. Rinse on large droplets of TBS-Tween for 6x2 min.

21. Post-Fixation: post-fix sections on droplets of 2% glutaraldehyde in 0.1M PB for 10 minutes and then rinse in TBS-Tween for 6x2 min. Note: this step may not be necessary.

22. Contrast Staining: rinse sections in 3x20 dips of distilled water, and then stain with uranyl acetate for 15 minutes and lead citrate for 1-2 minutes.

23. Observation: observe sections under electron microscopy.


Solutions and Reagents:


TBS-Tween (0.05M TBS, 0.05% Tween 20, pH 7.6):

Trizma base ------------------------------- 6.1 g

NaCl ------------------------------------------ 9 g

Dstilled water ------------------------------1000 ml

Mix to dissolve and adjust pH to 7.6 using 1N HCl and then add 0.5 ml Tween 20


0.1M Glycine in 0.1M PB:

To prepare 100 ml, add 0.75 g of glycine to 100 ml of 0.1M PB.


0.2M Sucrose Solution:

To prepare 100 ml, add 8 g of sucrose to 100 ml of 0.1M PB.    


Post-fixative (2% glutaraldehyde in 0.1M PB):

To prepare 100 ml, add 4 ml of 50% glutaraldehyde to 95 ml of 0.1M PB


L.R. White:

Kit from EMS


Blocking Buffer (1% BSA, 3% NS, 0.1% Fish Gelatin, 0.05% Sodium Azide in 0.05M TBS, pH 7.6):

To prepare 100 ml,

BSA ---------------------------------------- 1 g

Normal serum --------------------------- 3 ml

Cold fish gelatin ------------------------ 0.1 ml

Sodium azide --------------------------- 0.05 g

0.05M TBS, pH 7.6 ------------------- 100 ml

Stir to dissolve.


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 C.


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 C. Note: the mount of NaOH is very important. Too much will make solution cloudy.




1. Primary antibody should be about 10 times more concentrated than LM working dilution and Overnight incubation seemed to be better than 2 hours at room temperature.

2. If doing double labeling, you need to repeat from step 14 to 19. A different blocking buffer may be needed depending on species of 2nd antibody.

3. For double labeling, use small gold conjugated streptavidin (i.e. 6nm) first, and use large gold conjugated streptavidin (i.e. 10nm) last.

4. This protocol was tested successfully in January 20, 2004 on cell pellet fixed for 4 hours with 4% paraformaldehyde and immunostained with a rabbit antibody.