Cyanuric Chloride and N-Methyl Morpholine in Methanol as a Fixative for Polysaccharides

Abstract

Rat submandibular and sublingual gland fragments 2 mm thick were immersed in 0.5% (0.025 M) cyanuric chloride in anhydrous methanol containing 1% (0.1 M) N-methyl morpholine at 23–27 C. After 18–24 hr, the tissues were washed in methanol, cleared in xylene, embedded in paraffin and sectioned at 3–8 μ. These tissues were compared with those fixed in: (1) 10% aqueous cold neutral buffered formalin, (2) anhydrous cold methanol, (3) 0.5% (0.014 M) cetylpyridinium chloride in 10% neutral buffered aqueous formalin, and (4) 0.4% (0.015 M) 5-aminoacridine in 50% aqueous ethanol. Tissues fixed in cyanuric chloride were well preserved with little loss or alteration of tissue components, particularly of epithelial mucins and serous components, in contrast to the poor preservation seen with the other fixatives. Tissues were examined by phase-contrast or following staining with Alcian blue 8GX and colloidal iron methods for complex acidic carbohydrates, and, the periodic acid-Schiff method for carbohydrates rich in vicinal hydroxyl groups. Dyes such as orange G, aniline blue and phosphotungstic acid-hematoxylin, which do not color or are not specific for carbohydrates, were also used to reveal structural differences resulting from the various methods of fixation. The cyanuration technique is simple to perform, reproducible and safe.