The Production of Large, Epoxy-Embedded, 50 μ Sections by Precision Sawing; A Preliminary to Survey for Ultrathin Sectioning

Abstract

Tissue slices not more than 4 mm thick—including those containing mineralized bone—were fixed in buffered 6.25% glutaraldehyde. Parts of the specimen to be used for subsequent electron microscopy had to be within 1 mm of the surface to permit proper penetration of the second fixation in 1% OsO₄, therefore, if such parts were not originally so exposed, superfluous tissue was dissected away. After fixation and washing, a regular dehydration and embedding sequence was used, but with the time in individual fluids tripled, and 2 changes of absolute ethanol and 2 of undiluted resin added to insure thorough permeation. The resin mixture (containing Epon 812) was the hardest one normally used for ultra-thin sectioning. The blocks were cut at 50 μ on a Gillings-Bronwill thin-sectioning, hardtissue saw, which had been modified by the addition of supplementary water jets for cooling the diamond blade and the tissue block. Sections were examined microscopically in glycerol; a selected area was punched out with a 1 mm diameter leather punch, and the resulting disk was cemented to a preformed blank casting adapted to the chuck of the ultramicrotome. Tissue specimens presenting surfaces of 1.5 × 3 cm can be surveyed rapidly with this method. The use of a punch to obtain the disks for ultramicrotomy leaves the rest of the section undamaged.