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Principles & ProtocolsPrinciples - Negative StainingJump to ProtocolWhy Negative stain?Your sample will be subjected to a rather harsh environment in the electron microscope. First, an ultra high vacuum is needed to allow the electrons to travel freely through the microscope's column, and second, bombardment of the sample with high energy electrons tends to cause chemical changes in the specimens (radiation damage). However, proteins need water molecules to preserve their structural integrity, therefore something has to be done to counter the drying effects of the vacuum. The oldest approach discovered to this end, is to replace the water molecules with a salt. Using salts in which the counterion is a heavy metal provides not only protection against structural collapse, but also atoms that scatter electrons strongly, rendering biological samples easy to see. The term "negative stain" arises from the fact that heavy metal salts make the sample appear darker than the background.What should I try?There are many different compounds that can be tried as negative stains. The most common stain used for electron microscopy is uranyl acetate, since the uranium atoms produce a very strong contrast of the protein against the background. Other compounds commonmy tried are phosphotunstic acid (PTA), uranyl formate, ammonium molybdate, lead citrate, lead acetate, as well as commercial stains, such as Nano-Van and Nano-W (vanadate and tungsten salts respectively).ConditionsConcentration, and pH are just two of the parameters that can be varied with negative stains. Uranyl salts (acetate and formate) tend to be quite acidic, and precipitate if the pH is brought closer to neutrality. At lower concentrations they tend to produce a smaller grain, revealing finer details, but making the preparation of good samples somewhat unreliable. PTA can be used at neutral pH, but reacts with phosphate buffers, producing images of poor quality. Nano-Van and Nano-W are stains that produce a rather fine grain, and are sold at neutral pH, however, not all samples stain well with these. The bottom line is that several stains should be tried with a given sample, and the best one chosen from direct evidence.Is there more than one way to prepare negatively stained samples?Unfortunately there are many different ways to prepare the samples, these being stained or ice embedded. Negatively stained samples are usually prepared on continuous carbon film (which itself can be prepared in several ways), but there are other methods that might be more suitable for other samples. Double carbon sandwiches can be made, deep stained samples, stain films over holes, etc. Factors to take into account include the type of data that needs to be collected (i.e., images for a publications, showing the size and shape of the molecule, or faithful projection images for a 3-D reconstruction).Where do I get the negative stains to start?At the NYSBC we are making an effort to keep a decent supply of stains to be used by our affiliates. We have stocks of uranyl acetate, uranyl formate (this has to be prepared fresh before using), ammonium molybdate, lead citrate, Nano-Van and Nano-W. Please remember that heavy metal salts tend to be highly toxic and a lot of caution should be exercised when using them (unless you want to become a heavy atom derivative). Please let us know if there is a stain you would like to try.Protocols
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