Material and methods

Abstract

In our studies cats and rats were used. Over the years a variety of preparatory methods have been employed. The demonstration of axonal degeneration has not proved to depend critically on the methods of fixation and embedding beyond that of securing a general good preservation of the tissue. In part, fixation has been by perfusion, with 10% formalin containing a varying amount of sucrose and buffered with phosphate, the vascular system first having been briefly rinsed with Ringer’s solution. In most cases, the formalin fixative introduced by Holt and Hicks (1961) has been used. The procedure followed in the cat has been described elsewhere (Walberg 1963 a). In the rat, perfusion through the abdominal aorta has been found to be advantageous. This, in contrast to an approach through the thoracic aorta or the left ventricle, does not interfere with respiration up to the moment of penetration of the fixative. Probably, perfusion with a glutar-aldehyde fixative (Sabatini et al. 1963) will prove as adequate for degeneration studies as perfusion with formalin. In part we have fixed the tissues by direct immersion in 2% osmium tetroxide solution, essentially according to Palade (1952), Caulfield (1957) or Millonig (1962). Material from animals perfused with formaldehyde was always post-fixed in one of these solutions. The fixatives and the dehydrating media were used chilled. As an embedding material, methacrylate (butyl/methyl 9:1) was used in our earlier studies, but later it was replaced by Araldite (Webster et al. 1961).