Notes on Tissue Preparation
With few exceptions, the tissues photographed for this atlas were prepared in our laboratory in the Department of Cellular and Structural Biology in the University of Colorado School of Medicine, in Denver, Colorado. We have improved methods for obtaining large (1-mm square), flat, contaminant-free thin sections that permit photography of large fields of view at very low magnifications with the transmission electron microscope. In addition, we have devised a method that greatly simplifies mounting these large thin sections on Formvar-coated slot grids and permits the photographing of any field of view uninterrupted by the grid bars present on common support screens. Rather than present a detailed description of these methods here, we refer the reader to the following reference, in which the methods of specimen preparation employed in this atlas are described in considerable depth:
Moran, D. T., and Rowley, J. C. III: Biological specimen preparation for correlative light and electron microscopy. In Correlative Microscopy: Instrumentation and Methodology. Edited by M. A. Hayat. New York, Academic Press, 1986, pp. 1-22.
Fresh tissue samples were fixed in one of a variety of buffered paraformaldehyde-glutaraldehyde mixtures, post-fixed in buffered 2% osmium tetroxide, dehydrated in a graded acetone series, and infiltrated with (and embedded in) epoxy resin. Thick section (1 µm thick) for light microscopy and thin sections (500 to 800 Å thick) were cut with a diamond knife mounted in a Porter-Blum MT-2B ultramicrotome. For light microscopy, thick sections were stained with toluidine blue and photographed in a Zeiss Ultraphot II photomicroscope fitted with Zeiss planapochromatic lenses. For electron microscopy, sections were mounted on Formvar-covered “slot” grids, using a new and effective device, the Domino Rack (Sundance Technology, Denver, CO). Once mounted on the slot grids, thin sections were doubly stained with uranyl acetate and lead citrate, and photographed with a Philips EM-300 transmission electron microscope.
We found nerve tissue particularly difficult to preserve and are indebted to several investigators for providing us with well-fixed tissue blocks from various regions of the nervous system. Tissues for Plates 8-1, 8-2, and 8-4 were kindly sent to us by Dr. Cedric Raine of the Albert Einstein College of Medicine; tissues for Plate 8-5 were given to us by Dr. Stephen Roper of Colorado State University; and the negative for Plate 8-4 was generously offered to us by Dr. Tom Mehalick of our department. In addition, the elegant scanning electron micrograph of a macrophage engaged in phagocytosis of an erythrocyte that appears in Plate 4-1 (Figure A) was given to us by Dr. Keith R. Porter of the University of Maryland. We are most grateful to these investigators for supplying us with examples of their work that enrich this atlas and enhance its instructional value.