Summary
This study explores the effects of two types of fixative on chromatin. The first type (acrolein, glutaraldehyde) engenders a high degree of ultrastructural preservation. The other type are fixatives that are widely used in cytochemistry and cytogenetics (acetic acid, 3∶1 by vol. methanol-acetic acid, methanol alone, formaldehyde).
Lymphocytes of adult rats so-fixedin vitro were prepared for electron microscopy or microdensitometric evaluations of smears. Assessments were made of variations in their total protein, nuclear basic protein and DNA contents. DNA was determined both as Feulgen-positive material and by its binding of intercalating dyes (Methyl Green, specific for double-stranded polynucleotides).
Our results showed that some fixatives break up the chromatin organization by acting on particular components of chromatin fibres. They can thus be considered to be destructive agentsin situ. In addition, a revaluation of some aldehyde fixatives is proposed for both ultrastructural and cytochemical research.
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References
Bedi, K. S. &Goldstein, D. J. (1974) Cytophotometric factors causing apparent differences between Feulgen-DNA content of different leukocytes types.Nature, Lond. 251, 439–40.
Bernhard, W. (1969) A new staining procedure for electron microscopical cytology.J. Ultrastruct. Res. 27, 250–65.
Böyum, A. (1964) Separation of white blood cells.Nature, Lond. 204, 793–94.
Bowes, J. H. &Cater, C. W. (1966) The reaction of glutaraldehyde with proteins and other biological materials.J. R. microsc. Soc. 85, 193–200.
Brody, T. (1974) Histones in cytological preparations.Expl Cell. Res. 101, 255–63.
Diaz, M. (1972) Methyl Green staining and highly repetitive DNA in polytene chromosomes.Chromosoma 37, 131–8.
Dick, C. &Johns, W. (1968) The effect of two acetic acid containing fixatives on the histone content of calf thymus deoxyribonuclearprotein and calf thymus tissue.Expl Cell Res. 51, 626–32.
Fraschini, A. &Marinozzi, V. (1977) Critical analysis of the use of the acrolein-Schiff method as a possible DNA reaction.Histochemistry 50, 197–206.
Fraschini, A., Bolchi, F. &Biggiogera, M. (1980) Ultrastructural cytotopochemistry of chromatin: approaches via destructive deproteinizing.J. submicrosc. Cytol. 12, 121–36.
Gaub, J. (1976) Feulgen-Naphthol Yellow S cytophotometry of liver cells.Histochemistry 49, 293–301.
Haselkorn, R. &Doty, P. (1961) The reaction of formaldehyde with polynucleotides.J. Biol. Chem. 236, 2738–45.
Hayat, M. A. (1970)Principles and Techniques of Electron Microscopy. Vol. 1. pp. 35–107. New York: Van Nostrand Reinhold.
Hopwood, D. (1968) Some aspects of fixation by glutaraldehyde and formaldehyde.J. Anat., Lond. 103, 581–89.
Itikawa, O. &Ogura, Y. (1954) The Feulgen reaction after hydrolysis at room temperature.Stain. Technol. 29, 237–49.
Kiefer, R., Kiefer, G. &Sandritter, W. (1972) Feulgenhidrolyse kinetic in eu- und eterochromatin.Histochemie 30, 150–5.
Kjellstrand, P. T. T. &Anderssen, G. K. A. (1975) Histochemical properties of spermatozoa and somatic cells. I: Relations between Feulgen hydrolysis pattern and the composition of the nucleoproteins.Histochem. J. 7, 563–73.
Le Boutellier, P., Kinsky, R., Vujanovic, N., Duc, H. &Voisin, G. (1976) Morphological differences between thymus and bone marrow derived lymphocytes.Differentiation 6, 125–41.
Le Boutellier, P., Kinsky, R., Righenzi, S. &Voisin, G. (1978) Electron microscopic study of thymus and bone marrow derived mouse lymphocytes.Ann. Immun. 129, 635–51.
Luft, J. (1959) The use of acrolein as a fixative for light and electron microscopy.Anat. Rec. 133, 305–12.
Manfrediromanini, M. G., Formenti, D., Fraschini, A., Pellicciari, C., Redi, C. A. &Scherini, E. (1981) Some parameters for a cell cycle cytotopochemical study.Gegenbaurs morphol. Jahrb., Leipzig 127, 581–7.
Marinozzi, V. (1963) The role of fixation in electron staining.J. R. microsc. Soc. 81, 141–54.
Mayall, B. (1966) The detection of small differences in DNA content with microspectrophotometric techniques.J. Cell Biol. 31, 74A.
Mayall, B. &Mendelsohn, M. (1967) Chromatin and chromosome compaction and the stoichiometry of DNA staining.J. Cell Biol. 35, 88A.
Mayall, B. (1969) Deoxyribonucleic acid cytophotometry of stained human leukocytes. I. Differences among cell types.J. Histochem. Cytochem. 17, 249–57.
Millonig, G. &Marinozzi, V. (1968) Fixation and embedding in electron microscopy. InAdvances in Optical and Electron Microscopy (edited byBarer, R. &Cosslett, V.) Vol. 2. pp. 251–341. London: Academic Press.
Mittermayer, C., Madreiter, H., Lederer, B. &Sandritter, W. (1971) Differential acid hydrolysis of euchromatin and heterochromatin.Beitr. Path. 143, 157–65.
Nicolini, C. (1979)Chromatin structure and function. Vol. A. pp. 293–322. New York: Plenum Press.
Olins, D. &Wright, E. (1973) Glutaraldehyde fixation of isolated eucaryotic nuclei.J. Cell Biol. 59, 304–17.
Pellicciari, C., Manfredi Romanini, M. G., Casirola, G., Ippoliti, G. & Marini, G. (1976) Hydrolysis kinetics studies of the Feulgen reaction on different lymphocyte populations in man.Fifth Int. Congr. Histochem. Cytochem., Bucharest.
Pellicciari, C. &Fraschini, A. (1978) Methods of denaturation and renaturation of DNA in interphasic chromatin: cytochemical quantitative analysis by Methyl Green staining.Histochem. J. 10, 213–22.
Rasch, R. &Rasch, E. (1973) Kinetics of hydrolysis during the Feulgen reaction for deoxyribonucleic acid. A re-evaluation.J. Histochem. Cytochem. 21, 1053–65.
Retief, A. &Rüchel, R. (1977) Histones removed by fixation. Their role in the mechanism of chromosomal banding.Expl. Cell Res. 106, 233–7.
Reynolds, E. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy.J. Cell Biol. 17, 208–13.
Sabatini, D., Bensch, K. &Barrnett, R. (1962) New means of fixation for electron microscopy and histochemistry.Anat. Rec. 142, 274–90.
Sabatini, D., Bensch, K. &Barrnett, R. (1963) Cytochemistry and electron microscopy: the preservation of the cellular ultrastructure and enzymatic activity by aldehyde fixation.J. Cell Biol. 17, 19–58.
Sabatini, D., Miller, F. &Barrnett, R. (1964) Aldehyde fixation for morphological and enzyme histochemical studies with the electron microscope.J. Histochem. Cytochem. 12, 57–71.
Scott, J. (1967) On the mechanism of the Methyl Green-pyronine stain for nucleic acids.Histochemie 9, 30–46.
Stollar, D. &Grossman, L. (1962) The reaction of formaldehyde with denatured DNA: spectrophotometric, immunologic and enzymic studies.J. molec. Biol. 4, 31–8.
Sumner, A., Evans, H. &Buckland, R. (1973) Mechanism involved in the banding of chromosomes with Quinacrine and Giemsa. I. The effects of fixation in methanol-acetic acid.Expl. Cell Res. 81, 214–22.
Trump, B. F. &Ericsson, J. L. (1965) The effect of the fixative solution on the ultrastructure of cells and tissues. A comparative analysis with particular attention to the proximal convoluted tubule of the rat kidney.Lab. Invest. 14, 1245–51.
Trump, B. F. &Bulger, R. E. (1966) New ultrastructural characteristics of cells fixed in a glutaraldehyde-osmium tetroxide mixture.Lab. Invest. 15, 368–79.
Van Duijn, P. (1961) Acrolein-Schiff: a new staining method for proteins.J. Histochem. Cytochem. 9, 234–41.
Zetterberg, A. &Auer, G. (1969) Early changes in the binding between DNA and histones in human leukocytes exposed to phytoemagglutinin.Exp. Cell Res. 56, 122–6.
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Fraschini, A., Pellicciari, C., Biggiogera, M. et al. The effect of different fixatives on chromatin: Cytochemical and ultrastructural approaches. Histochem J 13, 763–779 (1981). https://doi.org/10.1007/BF01003288
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DOI: https://doi.org/10.1007/BF01003288