The most frequently used method for post-staining is a twostep procedure of staining with uranyl acetate (UA), followed by lead citrate. Uranyl acetate is used as an aqueous or alcoholic solution with a pH for the saturated solution in the range of 3.5 to 4.0. The addition of alcohol, especially methanol, increases the solubility (Hayat, 2000). Uranyl acetate strongly stains proteins as well as nucleic acids and phospholipids. When applied after the uranyl acetate staining, lead citrate (prepared according to Reynolds, 1963) will increase this contrast (Dykstra, 1992). Staining can be performed either manually or automatically, both techniques have their advantages. For manual staining, a grid is floated, sectionside down, on a drop of a uranyl acetate solution for 10 minutes. After blotting off the stain, the grid is rinsed thoroughly with water to remove any residual unbound stain. This first step is followed by 5 min. lead citrate staining, following the same procedure. The consumption of reagents is minimal, whereas the effort is relatively high.
Alternatively, poststaining can be automated. This ensures increased reproducibility and time saving, though the amount of reagents used is higher. Using the automated contrasting device EM AC20 (Leica Microsystems, Vienna) allows for simultaneous staining of up to 20 grids per run with no effort and a guarantee for safety, both for the environment and the user. Although UA is an excellent and well characterized stain, replacements are sought for, for several reasons.
When it needs to be handled as a powder, it is very toxic and carcinogenic if inhaled. Furthermore, also depleted uranyl acetate is considered a radioactive material, and hence subject to relevant regulations. Therefore, UA requires adequate storage and careful handling, which in turn increases cost for shipping and waste disposal. To minimize contact, the automated version with the AC20 is preferred by many users, in particular as readily prepared solutions are available, handling of solid UA can be avoided.
Two reagents described in literature as replacements for UA caught our attention: oolong tea extract (OTE) and Platinum Blue. Only very little data is published about them and the methods are not well known in the EM community. Therefore, we have tested both with manual contrasting and for the first time with the Leica EM AC20 instrument.
To allow a direct comparison of results from the different post-staining techniques, the same sample was used for all tests: Liver tissue freshly dissected from mice was fixed with 2.5 % glutaraldehyde in 100 mmol/l Soerensen phosphate buffer and post-fixed with 2.0 % osmium tetroxide. Pieces of tissue were dehydrated and embedded in Agar 100 epoxy resin and sectioned to a nominal thickness of 70 nm. Poststaining was performed as described below. Besides contrasting efficiency and comparability of the results with UA, a number of other important parameters were assessed.