Staining at elevated temperatures also contributes to precipitates due to evaporation of the stain solvent. Prolonged staining times (longer than 10–15 min for uranyl acetate and longer than 5–10 min with lead stains) can result in stain precipitates due to drying at the surface of the drops of stain. Old stains loose reactivity when exposed to light and the atmosphere. Problems related to low contrast and stain precipitation are often related to age of the stains. Phosphate groups may be involved in lead staining since use of phosphate buffers often enhances overall staining. Lead stains are highly alkaline and react with carbon dioxide and oxygen to form lead carbonate (PbNO 3), a highly insoluble, electron dense precipitate ( see Fig. Lead binds to negatively charged groups such as hydroxyl groups and areas which reacted with osmium tetroxide such as membranes. This is probably the reason that the post-staining sequence of uranyl acetate followed by lead citrate works so well. An important attribute of uranyl stains is that uranium acts as a mordant for lead stains. Uranyl acetate stains have a pH of 3.5–4 and strongly stain proteins. Uranium reacts strongly with phosphate and amino groups to stain nucleic acids and phospholipids in membranes. Many stains are general, nonspecific stains however, certain chemical groups react with uranium and lead ions. The most common staining method in biological TEM consists of post-staining sections on grids with aqueous uranyl acetate followed by Reynolds’ lead citrate. There are numerous methods in the literature and various commercial devices for post-staining grids the methods presented here are based on keeping the procedures simple and reproducible and use common laboratory materials. A number of other lead stains were used in the early 1960s however, the most reliable lead stain has been the lead citrate stain published by Reynolds in 1963. Watson published on the use of uranium salts to stain tissue sections and introduced the use of lead stains. The grids were then washed on drops of distilled water. Gibbons and Bradfield first used aqueous solutions of lanthanum nitrate and osmium tetroxide in a simple procedure for staining sections on grids for TEM which consisted of floating grids, section side down, in drops of stain. Heavy metal stains such as uranium (atomic number 92), lead (atomic number 82), and osmium (atomic number 76) have been used extensively as electron dense stains. Factors that affect contrast in biological TEM are section thickness, the presence of heavy atoms in the specimen, accelerating voltage, and aperture size. There is little inherent difference in contrast between embedding media and biological specimens since they are chemically similar in consisting of carbon, hydrogen, oxygen, and other transparent (non-electron dense) atoms ( see Fig. Staining of biological tissues is needed to improve differential contrast of specimens imaged in the transmission electron microscope (TEM). In addition, stain precipitates on grids often can be removed by treatment with 10 % (v/v) acetic acid. Marinozzi rings and microwave-assisted post-staining offer alternatives to traditional grid staining. All of these stains are compatible with aqueous fixatives and should be considered when the usual stains are not satisfactory. Tannic acid and PPD improve membrane preservation, and malachite green is a phospholipid stain. Tannic acid, paraphenylenediamine (PPD), and malachite green can all serve as en bloc stains and can contribute to overall improved visualization of ultrastructural details in biological specimens. Over the years, several other treatments have been developed for use with the primary fixation or during dehydration. These procedures can be as simple as en bloc staining with uranyl acetate after primary fixation and osmication. When it is apparent that simple, aqueous uranium and lead post-staining is not adequate, other stains are invoked. The most common post-staining of sections is done on grids by aqueous uranyl acetate followed by lead citrate. Often specimens are fixed and stained with osmium tetroxide during fixation, but additional contrast is the result of additional heavy metal stains on the sections. Post-staining of ultrathin sections and/or en bloc staining of specimens is necessary for differential contrast and improved resolution of cellular structures.
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