"Sato's Lead" Recipe

A popular stain that has been modified over the years

This stain, often times called "Sato's Lead" or "Sato's Staining Procedure" is used widely as a routine stain for TEM thin sections. Lead solutions are often times used after uranyl staining to yield high contrast and to stain many cellular and tissue components.

The basic staining procedure was first published by T. Sato, J. of Electron Microscopy 17, 2 , 158 (1968). The method was in fact surely influenced by earlier work published by Watson in 1958.

Other references for other variations:
Karnovsky, M. J., "Simple methods for 'staining with lead' at high pH in electron microscopy," J. Biochem. Biophys. Cytol. 11, 729 (1961).

Millonig G., "A modified procedure for lead staining of thin sections", J. Biophys. Biochem Cytol 11, 736 (1961).

Reynolds, E. S., "The use of lead citrate at high pH as an electron-opaque stain in electron microscopy", J.Cell Biol.17, 208 (1963).

Venable, J. H., and Coggeshall, R., "A simplified lead citrate stain for use in electron microscopy", J.Cell Biol.25, 407 (1965).

Fahmy, A., "An extemporaneous lead citrate stain for electron microscopy", Proc. 25th Ann. Conf. EMSA, p.148 (1967).

Sato T, "A modified method for lead staining of thin sections", J. Electron Microsc., 16:133 (1967).

The original recipe was as follows:

1gm lead nitrate

1gm lead acetate

1gm lead citrate

2gms sodium citrate

Dissolve in 82 ml distilled water, then add 18 ml 4% NaOH, pH of 12. If the solution has a milky color to it, then everything is going properly! Solution should be stored refrigerated.

For use, add 18 ml freshly prepared (from pellets) NaOH (1N). The solution will clear except for some large grains or precipitate at the bottom. Filter rapidly and store, sealed in dark bottle, plastic recommended, not glass.

A more modern version of this recipe/protocol is the following:

0.20 gm anhydrous lead citrate Pb(C₆H₅O₇)₂ (FW = 1053.85)*

0.15 gm lead nitrate

Pb(NO₃)

0.15 gm lead acetate

Pb(CH₃COO)₂·3H₂O 1.00 gm sodium citrate Na₃

(C₆H₅O₇)·2H₂O 41.0 ml distilled water

* we suggest substituting with SPI's lead citrate trihydrate - 0.21g FW = 999.85)

Above recipe taken from the following publication:
Hanaichi, T., Sato, T, Hoshino, M., and Mizuno, N., Proc. XI Congress on Electron Microscopy (ICEM), Kyoto 1986.

Above ingredients are placed in a 50 ml volumetric flask and mixed well to a yellowish milky solution. Then the solution is added to 9.0 ml of 1 N NaOH and mixed well until the solution becomes transparent with light yellowish color. Always store refrigerated. Useful shelf life of the mixed solution could be on the order of one year.

For further reading on other references to the procedure:

1. Journal of Electron Microscopy 116, 133 (1976)

2. Hanaichi, T., et. al., A Stable Lead by Modification of Sato's Method, J . Electron Microscopy 35, (304-306) 1986.

3. Takagi, I., et. al., Penetration of Stainability of Modified Sato's Lead Staining Solution, J. Electron Microscopy 39, (67-68) 1990.

Actual use observations:
When ready to use, if a white precipitate has formed on the top of the solution, remove staining solution away from the precipitate. It is also permissible to microfuge just before use to remove the precipitate. Lead carbonate precipitate has the appearance of very black circles (about 100- 200 nm) with one side usually rough or indented. If the precipitate is more like a "pepper", that is, smaller in size, then it is probably uranyl acetate, not the carbonate. Phosphate buffers can also look like "pepper" as well, but the grains are usually somewhat darker. Uranyl acetate deposits are more often than not on subcellular structures, for example, membranes, ribosomes, etc.; phosphate deposits are usually in the cytosol, where you would expect water.

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