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. 1967 Sep 1;34(3):757–771. doi: 10.1083/jcb.34.3.757

ULTRATHIN FROZEN SECTIONS

I. Methods and Ultrastructural Preservation

W Bernhard 1, Elizabeth H Leduc 1
PMCID: PMC2107175  PMID: 4167504

Abstract

A relatively simple method for obtaining ultrathin, frozen sections for electron microscopy has been developed. Tissues, cultured cells, and bacteria may be employed. They are fixed in 1.25–4% glutaraldehyde for 1–4 hr, are washed overnight in buffer at 3°C, and are embedded in 20% thiolated gelatin or pure gelatin. Before sectioning they are partially dehydrated in 50% glycerol, frozen in liquid nitrogen on a modified tissue holder, and subsequently maintained at -70°C with dry ice. Finally, they are sectioned very rapidly with glass knives on a slightly modified Porter-Blum MT-1 microtome in a commercial deep-freeze maintained at -35°C and are floated in the trough of the knife on a 40% solution of dimethylsulfoxide (DMSO). The sections are picked up in plastic loops and transferred to distilled water at room temperature for thawing and removal of the DMSO, placed on grids coated with Formvar and carbon, air-dried, and stained with phosphotungstic acid, sodium silicotungstate, or a triple stain of osmium tetroxide, uranyl acetate, and lead. Large flat sections are obtained in which ultrastructural preservation is good. They are particularly useful for cytochemical studies.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BERNHARD W. ULTRAMICROTOMIE 'A BASSE TEMP'ERATURE. Annee Biol. 1965 Jan-Feb;41:5–19. [PubMed] [Google Scholar]
  2. FERNANDEZ-MORAN H., FINEAN J. B. Electron microscope and low-angle x-ray diffraction studies of the nerve myelin sheath. J Biophys Biochem Cytol. 1957 Sep 25;3(5):725–748. doi: 10.1083/jcb.3.5.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. FERNANDEZ-MORAN H. Low-temperature preparation techniques for electron microscopy of biological specimens based on rapid freezing with liquid helium II. Ann N Y Acad Sci. 1960 Apr 13;85:689–713. doi: 10.1111/j.1749-6632.1960.tb49990.x. [DOI] [PubMed] [Google Scholar]
  4. GERSH I., ISENBERG I., STEPHENSON J. L., BONDAREFF W. Submicroscopic structure of frozen-dried liver specifically stained for electron microscopy. Anat Rec. 1957 May;128(1):91–111. doi: 10.1002/ar.1091280109. [DOI] [PubMed] [Google Scholar]
  5. GILEV V. P. The use of gelatin for embedding biological specimens in preparation of ultrathin sections for electron microscopy. J Ultrastruct Res. 1958 Aug;1(4):349–358. doi: 10.1016/s0022-5320(58)90006-6. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Leduc E. H., Bernhard W. Recent modifications of the glycol methacrylate embedding procedure. J Ultrastruct Res. 1967 Jul;19(1):196–199. doi: 10.1016/s0022-5320(67)80068-6. [DOI] [PubMed] [Google Scholar]
  8. Leduc E. H., Holt S. J. Hydroxypropyl methacrylate, a new water-miscible embedding medium for electron microscopy. J Cell Biol. 1965 Jul;26(1):137–155. doi: 10.1083/jcb.26.1.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. MARINOZZI V. CYTOCHIMIE ULTRASTRUCTURALE DU NUCL'EOLE--RNA ET PROT'EINES INTRANUCL'EOLAIRES. J Ultrastruct Res. 1964 Jun;10:433–456. doi: 10.1016/s0022-5320(64)80021-6. [DOI] [PubMed] [Google Scholar]
  10. MUNDKUR B. ELECTRON MICROSCOPICAL STUDIES OF FROZEN-DRIED YEAST. V. LOCALIZATION OF PROTEIN-BOUND SULPHYDRYL. Exp Cell Res. 1964 Mar;34:155–181. doi: 10.1016/0014-4827(64)90192-2. [DOI] [PubMed] [Google Scholar]
  11. MUNDKUR B. Electron microscopical studies of frozen-dried yeast. I. Localization of polysaccharides. Exp Cell Res. 1960 Jun;20:28–42. doi: 10.1016/0014-4827(60)90219-6. [DOI] [PubMed] [Google Scholar]
  12. MUNDKUR B. Electron microscopical studies of frozendried yeast. II. The nature of basophile particles and vesicular nuclei in Saccharomyces. Exp Cell Res. 1961 Oct;25:1–23. doi: 10.1016/0014-4827(61)90303-2. [DOI] [PubMed] [Google Scholar]
  13. NELSON L. Cytochemical studies with the electron microscope. I. Adenosinetriphosphatase in rat spermatozoa. Biochim Biophys Acta. 1958 Mar;27(3):634–641. doi: 10.1016/0006-3002(58)90398-6. [DOI] [PubMed] [Google Scholar]
  14. NELSON L. Cytochemical studies with the electron microscope. II. Succinic dehydrogenase in rat spermatozoa. Exp Cell Res. 1959 Feb;16(2):403–410. doi: 10.1016/0014-4827(59)90269-1. [DOI] [PubMed] [Google Scholar]
  15. Pease D. C. The preservation of unfixed cytological detail by dehydration with "inert" agents. J Ultrastruct Res. 1966 Feb;14(3):356–378. doi: 10.1016/s0022-5320(66)80054-0. [DOI] [PubMed] [Google Scholar]
  16. SHELDON H., ZETTERQVIST H., BRANDES D. Histochemical reactions for electron microscopy: acid phosphatase. Exp Cell Res. 1955 Dec;9(3):592–596. doi: 10.1016/0014-4827(55)90092-6. [DOI] [PubMed] [Google Scholar]
  17. SJOSTRAND F. S., BAKER R. F. Fixation by freezing-drying for electron microscopy of tissue cells. J Ultrastruct Res. 1958 Apr;1(3):239–246. doi: 10.1016/s0022-5320(58)80005-2. [DOI] [PubMed] [Google Scholar]

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