Hot Alcoholic Phosphotungstic Acid and Uranyl Acetate as Routine Stains for Thick and Thin Sections

One of the potential advantages of high-voltage electron microscopes is that their greater penetrating power allows the use of thick sections (0.22 lt) to study three-dimensional cytoarchitecture . So far, this advantage has been limited because conventional methods of staining only allow the penetration of heavy metal solutions into sections to a depth of a few hundred Angstroms . For example, Glauert and Mayo (1970), using a 1 Mev microscope, say " . . . for sections thicker than 0 .5 μm stains must be introduced into the section before embedding, since stains will not penetrate the sections, even if the time of staining is prolonged ." The problem has been partly solved by staining tissues with hot uranyl acetate before embedment (4) but this treatment only gives marginally adequate contrast at 650 kv (Fig . 11) . The object of the work now reported was to find a way by which heavy metal solutions could penetrate tissues already embedded in plastic . A simple method is described for staining material already in resin uniformly to depths of 15 μ to give excellent contrast even for accelerating voltages of 650 kv . The procedure promises to allow the study of biological material with the high-voltage electron microscope in a way not possible hitherto. For thin sections at 100 kv it gives very high contrast uniformly through the section without surface precipitation . The procedure is easy and repeatable . It has also been possible to stain sections embedded in resins which have proved refractory to staining by other methods .