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Part IV. Morphological Procedures
Part IVI Morphological Procedures Electron-microscopic immunocytochemical procedures appear to be reaching their maturity. Nevertheless, as Wright and Rine point out (Chap. 23, this volume), no one procedure is suitable for all antigenantibody pairs. Despite the anatomic precision of methods using colloidal gold, the stoichiometry of antigen detection is certainly not quantitative. “Preembedding” procedures with peroxidase amplification, though qualitative, appear to be the most sensitive. In addition to the procedures described in the following chapters, frozen sucrose embedding procedures (Griffiths et al., 1983; Keller et al., 1984; see also the references cited by Bergmann, Part I, “Methods in Cell Biology,” Vol. 32) and procedures based on the LR hydrophilic resins of Polysciences (Newman et al., 1983) have had striking success. Protocols for “preembedding” immunocytochemistry (and immunofluorescence) are also described in Chapter 10 by Kuismanen and Saraste in Volume 32 of this series. Other important electron-microscopic procedures are those concerned with the representative sampling of isolated subcellular fractions either for anatomic study or immunocytochemical work (Baudhuin et al., 1967; Tartakoff and Jamieson, 1974; DeCamilli et al., 1983; Palade et al., 1983), procedures for rotary replication of etched samples (Heuser, 1981), and three-dimensional imaging (Turner, 198 1). What is equally impressive is the increased sophistication of light and fluorescence microscopy, as exemplified in Volumes 29 and 30 of this series, which describe fluorescent procedures. (See Contents of Recent Volumes for their Tables of Contents.) An outstanding example of the use of fluorescence microscopy is for intracellular pH determination, an issue corresponding to Chapter 22 on electron-microscopic immunocytochemistry by Anderson, and related to the chapters of Wilson and Murphy (Chap. 16, this volume) and Schindler et al. (Chap. 18, Vol. 32). Chapter 20 by Bacallao and Stelzer on confocal fluorescence microscopy illustrates a particularly dramatic technical advance that is still under development. Chapter 19 by Pringle et al. contains a wealth of practical advice of importance for the study of any cell type-not only yeast. 355
REFERENCES Baudhuin, P., Evrard, P., and Berthet, J. (1967). Electron microscopic examination of subcellular fractions. J . Cell Biol. 32, 181-191. DeCamilli, P., Hams, S . , Huttner, W., and Greengard, P. (1983). Synapsin I, a nerve terminal-specific phosphoprotein 11: Its specific association with synaptic vesicles demonstrated by immunocytochemistry in agarose-embedded synaptosomes. J . Cell Biol. 96, 1355-1373. Griffiths, G., Simons, K., Warren, G., and Tokuyasu, K. (1983). Immunoelectron microscopy using thin, frozen sections: Application to studies of the intracellular transport of semliki forest virus spike glycoproteins. I n “Methods in Enzymology” (S. Fleischer and B. Fleischer, eds.), Vol. 96, pp. 466-484. Academic Press, New York. Heuser, J. (1981). Preparing biological samples for stereomicroscopy by the quick-freeze, deep-etch, rotary-replication technique. I n “Methods in Cell Biology” (J. N. Turner, ed.), Vol. 22, pp. 97-122. Academic Press, New York. Keller, G.-A., Tokuyasu, K., Dutton, A., and Singer, S. J. (1984). An improved procedure for immunoelectron microscopy: Ultrathin plastic embedding of immunolabeled ultrathin frozen sections. Proc. Natl. Acad. Sci. U . S . A . 81, 5744-5747. Newman, G., Jasani, B., and Williams, E. D. (1983). A simple post-bedding system for a rapid demonstration of tissue antigens under the electron microscope. Histochem. J . 15, 543. Palade, P., Saito, A,, Mitchell, R., and Fleischer, S. (1983). Preparation of representative samples of subcellular fractions for electron microscopy by filtration with dextran. J . Histochem. Cytochem. 31, 971-974. Tartakoff, A,, and Jamieson, J. (1974). Subcellular fractionation of the pancreas. I n “Methods in Enzymology” ( S . Fleischer and L. Packer, eds.), Vol. 31, pp. 41-59. Academic Press, New York. Turner, J. N. (1981). “Methods in Cell Biology,” Vol. 22. Academic Press, New York.
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REFERENCES Baudhuin, P., Evrard, P., and Berthet, J. (1967). Electron microscopic examination of subcellular fractions. J . Cell Biol. 32, 181-191. DeCamilli, P., Hams, S . , Huttner, W., and Greengard, P. (1983). Synapsin I, a nerve terminal-specific phosphoprotein 11: Its specific association with synaptic vesicles demonstrated by immunocytochemistry in agarose-embedded synaptosomes. J . Cell Biol. 96, 1355-1373. Griffiths, G., Simons, K., Warren, G., and Tokuyasu, K. (1983). Immunoelectron microscopy using thin, frozen sections: Application to studies of the intracellular transport of semliki forest virus spike glycoproteins. I n “Methods in Enzymology” (S. Fleischer and B. Fleischer, eds.), Vol. 96, pp. 466-484. Academic Press, New York. Heuser, J. (1981). Preparing biological samples for stereomicroscopy by the quick-freeze, deep-etch, rotary-replication technique. I n “Methods in Cell Biology” (J. N. Turner, ed.), Vol. 22, pp. 97-122. Academic Press, New York. Keller, G.-A., Tokuyasu, K., Dutton, A., and Singer, S. J. (1984). An improved procedure for immunoelectron microscopy: Ultrathin plastic embedding of immunolabeled ultrathin frozen sections. Proc. Natl. Acad. Sci. U . S . A . 81, 5744-5747. Newman, G., Jasani, B., and Williams, E. D. (1983). A simple post-bedding system for a rapid demonstration of tissue antigens under the electron microscope. Histochem. J . 15, 543. Palade, P., Saito, A,, Mitchell, R., and Fleischer, S. (1983). Preparation of representative samples of subcellular fractions for electron microscopy by filtration with dextran. J . Histochem. Cytochem. 31, 971-974. Tartakoff, A,, and Jamieson, J. (1974). Subcellular fractionation of the pancreas. I n “Methods in Enzymology” ( S . Fleischer and L. Packer, eds.), Vol. 31, pp. 41-59. Academic Press, New York. Turner, J. N. (1981). “Methods in Cell Biology,” Vol. 22. Academic Press, New York.
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