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SIMPLE METHODS FOR "STAINING WITH LEAD" AT HIGH PH IN ELECTRON MICROSCOPY

M. Karnovsky
Published 1961 · Biology, Medicine

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The lead hydroxide stain of Watson (1958) used for increasing contrast in thin sections for electron microscopy has found acceptance in many laboratories. However, this stain has an unfortunate tendency to form precipitates (probably of lead carbonate (5)) on exposure to the air, thus contaminating the sections and irritating the observer. This drawback has led to the development of several modifications (2, 3) of the original method of staining and the use of ingenious devices (4, 5) for preventing exposure to air and consequent precipitate formation. We offer the following alternative methods which, we believe, are simpler to perform than those hitherto described. They have the additional advantages mentioned below. The methods are based on the observation that highly alkaline solutions of lead salts (pH > 11.5) yield relatively stable solutions which stain rapidly and intensely, thus obviating the hazard of precipitation to a marked degree. The methods have these additional advantages: the staining solutions are easily and rapidly prepared, are simply stored, and are stable for long periods of time. Furthermore, they can be efficiently used, many grids being treated simultaneously, without excessive precautions being taken against lead carbonate precipitation. Finally, "difficult" material, embedded in media which characteristically yield rather low contrast, such as epoxide resins, can be rapidly and easily stained. "C lean" preparations, of high contrast, are routinely obtained. As will be discussed later, it is thought that in these highly alkaline staining solutions lead is present as an hydroxide complex anion (plumbite ion) and that this anion is responsible for the staining. The methods of preparation are based on this hypothesis. Two methods for preparing the staining solutions have been found useful:
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