Ultracryotomy: A routine procedure - Iglesias, J.R.; Bernier, R.; Simard, R. (Elsevier)
A step further has been accomplished in obtaining ultrathin frozen sections routinely with a commercial ultramicrotome (Reichert OMU"2-Fc 150). A variety of animal tissues has been used as test materials with a standard fixation of 2.5% cacodylate-buffered glutaraldehyde. After fixation, the tissue is embedded in gelatin or simply supported by a drop of methyl cellulose 3% on the specimen holder to be immediately frozen by immersion in liquid nitrogen. With the specimen holder still immersed in liquid nitrogen, the piece of tissue is trimmed with a nitrogen-cooled blade to obtain a perfect pyramid. Ultrathin frozen sections are cut with the thermic advance of the ultramicrotome, using 50% dimethyl sulfoxide (DMSO) in distilled water as flotation liquid. Other substances have been tried. A wide range of temperatures for the specimen and for the knife have been tested: -50^oC for the knife and -70^oC for the specimen have been found to give best results. The sections are collected on a Formvarand-carbon coated hydrophilic grids or with small plastic rings and air-dried.Three staining procedures have been used: phosphotungstic acid (PTA) at pH 7.3, PTA at pH 1.0, and uranyl acetate 0.5% followed by lead citrate. The ultrastructural preservation and two applications (acid PTA as a stain for complex carbohydrates and alkaline phosphatase localization directly on ultrathin sections) are presented. It was also possible to prepare ultratbin frozen sections of unfixed tissue.The advantages and limitations of the technique, its present applications and future possibilities are discussed in detail.
http://dx.doi.org/10.1016/S0022-5320(71)80104-1
Keywords: W