Procedure:

1.   Refer to routine TEM protocol as needed. 

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

3.   Collect sections on formvar-carbon coated nickel grids, and allow the grids to dry overnight prior to staining.

4.   Pretreatment: staining tray filled with antigen retrieval solution (based on LM test, i.e., citrate buffer pH 6.0). Place grids into staining tray with tissue side face up, then put the tray in pre-heated steamer for 30 minutes, and allow grids to cool for 20 minutes.

5.   Rinse sections for 2x2 min with PBS-Tween 20

6.   Serum Blocking: incubate grids in normal serum blocking solution (species match secondary antibody) for 1-2 hours.

7.   Primary Antibody: incubate grids in primary antibody diluted in primary antibody dilution buffer for 1 hour at room temperature and the overnight at 4 ºC, then room temperature for another hour. Note: primary antibody dilution is usually 10 times more concentrated than immunostaining at LM level.

8.   Rinse sections with PBS-Tween20 for 3x2 min.

9.   Secondary Antibody: incubate grids in biotinylated secondary antibody (1:50, Vector Lab) in PBS for 2 hour at room temperature.

10.  Rinse with PBS-Tween20 for 3x2 min.

11.  Streptavidin-Gold: incubate grids with 10nm gold conjugated streptavidin (1:20, EMS) in PBS, pH 7.2 for 2 hours. 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.

12.  Rinse with PBS-Tween20 for 3x2 min.

13.  Rinse in distilled water. If time allowed, continue the following steps or let the grids air dry and do contrast staining later.

14.  Contrast Staining: stain with uranyl acetate for 15 minutes and lead citrate for 2-5 minutes.

15.  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

0.2M Sucrose Solution:

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

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

Notes:

1. Primary antibody and secondary 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.

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 on formalin fixed paraffin section re-embedded into EMbed-812 block and stained with a mouse antibody.