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.

Notes:

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 2^nd 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.