By Lawrence M. Schwartz and Barbara A. Osborne (Eds.)
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Add 2 g citric acid. Dissolve, filter, and make up to 1 liter with distilled water. 1 g Eosin Y 100 ml 1 : 1 Glycerol and methanol Dissolve and filter. 4 with hydrochloric acid. John F. R. Kerr 26 et al. Table IV Fixing and Processing Specimens for Light Microscopy Step 1. Fixation 2. Dehydration 3. Clearing 4. Wax infiltration 5 . 2 70% Ethanol LR grade 95% Ethanol LR grade 100% Ethanol LR grade Xylol Paraplast wax (MP 57-58C) Embed tissue in molten paraplast wax 24-48 hr 1 x 1hr 2xlhr 3xlhr 2xlhr 2 x 1 hrat60"C with a low nucleus :cytoplasm ratio.
VOL. 46 Copyright 8 1995 by Academic Press. Inc. All rights of reproduction in any form reserved 41 42 Alan Eastman investigators have observed apoptosis with no detectable internucleosomal DNA digestion. However, Arrends et al. (1990) demonstrated that injection of an endonuclease into nuclei produced apoptotic morphology, suggesting that the endonuclease activity is an essential event and not just the product of cell death. The discrepancy is resolved by the realization that DNA digestion to nucleosome-length fragments is probably overkill and that even a few breaks into high molecular weight fragments are adequate to produce the morphological changes associated with apoptosis.
Moreover, as stated earlier, PCD is coupled with dead cell elimination by convenient routes. In contrast, necrosis is typified by the in situ accumulation of the dead corpuscles; their evacuation takes much longer. Consequently, a necrotic site might occasionally display numerous nuclear fragments that are also TUNEL positive, yet the nature of the tissue damage cannot be mistaken for PCD. VIII. Perspectives PCD adds a new dimension to the biology of multicellular organisms. The entire cell becomes the elementary unit of tissue remodeling and cross-talk occurs between cells in a binary language of all or none.