Electron dense artefactual deposits in tissue sections: the role of ethanol, uranyl acetate and phosphate buffer

The occurrence of electron dense deposits in sections of aldehyde-fixed tissue prepared for transmission electron microscopy has been attributed to a number of conflicting factors. In an attempt to clarify this, the precipitating effect of different combinations of phosphate or cacodylate buffer, glutaraldehyde, ethanol and uranyl acetate was investigated in test tubes. As a preliminary investigation the combination of phosphate buffer, ethanol and uranyl acetate was investigated in heart and kidney tissue fixed in glutaraldehyde with or without postosmication. The essential factors in the formation of electron dense deposits in these tissues appear to be phosphate buffer, ethanol, and uranyl acetate, although glutaraldehyde may contribute in some way. The nature and intensity of the deposits seem to vary with the sequence of combination of these factors. Osmium did not appear to be an essential factor in the reaction since deposits were observed in both osmicated and unosmicated tissue. To avoid such deposits, a postosmication distilled water wash for 20 to 30 min followed by en bloc staining with aqueous uranyl acetate is advised if phosphate buffer is used as a fixative vehicle or buffer wash after the primary fixative.