Abstract
Ultrathin frozen sections of fresh liver tissue were floated on actinomycin D-3H. Quantitative high resolution radioautography was performed to determine the value of the method for detection of DNA by electron microscopy. A complete series of control experiments involving various treatments of frozen sections with enzymes (pronase, DNase) and 0.1 N HCl were also carried out to determine the specificity of the labeling. The results indicate the value of the method for detection of DNA directly on ultrathin frozen sections. Short treatments with pronase followed by DNase reduce the labeling to zero, whereas removal of chromosomal proteins with HCl increases the amount of radioactivity in the nucleus considerably. The results are discussed in view of the future applications opened by ultracryotomy, since radioautographic detection of various macromolecules and cellular components by labeled compound with specific affinities will now be possible.
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Selected References
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- BRACHET J., FICQ A. BINDING SITES OF 14C-ACTINOMYCIN IN AMPHIBIAN OVOCYTES AND AN AUTORADIOGRAPHY TECHNIQUE FOR THE DETECTION OF CYTOPLASMIC DNA. Exp Cell Res. 1965 Apr;38:153–159. doi: 10.1016/0014-4827(65)90437-4. [DOI] [PubMed] [Google Scholar]
- Beato M., Seifart K. H., Sekeris C. E. The effect of cortisol on the binding of actinomycin D to and on the template activity of isolated rat liver chromatin. Arch Biochem Biophys. 1970 May;138(1):272–284. doi: 10.1016/0003-9861(70)90308-5. [DOI] [PubMed] [Google Scholar]
- Bernhard W., Leduc E. H. Ultrathin frozen sections. I. Methods and ultrastructural preservation. J Cell Biol. 1967 Sep;34(3):757–771. doi: 10.1083/jcb.34.3.757. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bernhard W., Viron A. Improved techniques for the preparation of ultrathin frozen sections. J Cell Biol. 1971 Jun;49(3):731–746. doi: 10.1083/jcb.49.3.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bolund L. Actinomycin D binding to isolated deoxyribonucleoprotein and intact cells. Exp Cell Res. 1970 Nov;63(1):171–188. doi: 10.1016/0014-4827(70)90346-0. [DOI] [PubMed] [Google Scholar]
- Brachet J., Hulin N. Binding of tritiated actinomycin and cell differentiation. Nature. 1969 May 3;222(5192):481–482. doi: 10.1038/222481a0. [DOI] [PubMed] [Google Scholar]
- Camargo E. P., Plaut W. The radioautographic detection of DNA with tritiated actinomycin D. J Cell Biol. 1967 Dec;35(3):713–716. doi: 10.1083/jcb.35.3.713. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Iglesias J. R., Bernier R., Simard R. Ultracryotomy: a routine procedure. J Ultrastruct Res. 1971 Jul;36(1):271–289. doi: 10.1016/s0022-5320(71)80104-1. [DOI] [PubMed] [Google Scholar]
- Leduc E. H., Bernhard W., Holt S. J., Tranzer J. P. Ultrathin frozen sections. II. Demonstration of enzymic activity. J Cell Biol. 1967 Sep;34(3):773–786. doi: 10.1083/jcb.34.3.773. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Leduc E. H., Scott G. B., Avrameas S. Ultrastructural localization of intracellular immune globulins in plasma cells and lymphoblasts by enzyme-labeled antibodies. J Histochem Cytochem. 1969 Apr;17(4):211–224. doi: 10.1177/17.4.211. [DOI] [PubMed] [Google Scholar]
- Ringertz N. R., Darzynkiewicz Z., Bolund L. Actinomycin binding properties of stimulated human lymphocytes. Exp Cell Res. 1969 Aug;56(2):411–417. doi: 10.1016/0014-4827(69)90032-9. [DOI] [PubMed] [Google Scholar]
- Steinert G., Van Gansen P. Binding of 3H-actinomycin to vitelline platelets of amphibian oocytes. A high resolution autoradiographic investigation. Exp Cell Res. 1971 Feb;64(2):355–365. doi: 10.1016/0014-4827(71)90088-7. [DOI] [PubMed] [Google Scholar]
