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: