A Guide to Fracture Label: Cytochemical Labeling of Freeze-Fractured Cells

  • Pedro Pinto da Silva
  • M. L. F. Barbosa
  • A. P. Aguas


Freeze etching of biological specimens was devised to observe platinum/ carbon (Pt/C) casts of virus crystals frozen and crushed at low temperature (Steere 1957). The method was perfected by Moor and co-workers (1961), who modified high-vacuum equipment to allow “cutting” of specimens at controlled temperatures and at the low pressure required to obtain Pt/C replicas of high resolution. As designed by Moor and coworkers (1961), the freeze-etching apparatus was envisaged as a “freezing ultramicrotome” that produced a cut face of a frozen preparation. “Cutting” was followed by a period of sublimation (“etching”) thought necessary to reveal details of the microanatomy of cells (Moor et al. 1961; Moor and Mühlethaler 1963; Branton and Moor 1964). The first “freeze-fracture’ study was performed by Branton (1966), paradoxically still called “freeze etching” with “no etching.” For years, many researchers kept on submitting their freeze-frac-tured preparations to a period of “etching.” This procedure failed to reveal additional details because the specimens were impregnated in glycerol.


Colloidal Gold Wheat Germ Agglutinin Freeze Fracture Boar Sperm Cell BioI 
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  1. Aguas AP, Pinto da Silva P (1983) Regionalization of transmembrane glycoproteins in the plasma membrane of boar sperm head is revealed by fracture-label. J Cell Biol 97:1356–1364PubMedCrossRefGoogle Scholar
  2. Aguas AP, Pinto da Silva P (1984) High density of transmembrane glycoproteins on the flagellar surface of boar sperm cells. J Cell Biol 99:655–660PubMedCrossRefGoogle Scholar
  3. Aguas AP, Pinto da Silva P (1985) The acrosomal membrane of boar sperm: a Golgi-derived membrane poor in glycoconjugates. J Cell Biol 100:528–534PubMedCrossRefGoogle Scholar
  4. Barbosa MLF, Pinto da Silva P (1983) Restriction of glycolipids to the outer half of a plasma membrane: Concanavalin A labeling of membrane halves of Acanthamoeba castellanii. Cell 33:959–966PubMedCrossRefGoogle Scholar
  5. Bearer EL, Friend DS (1980) Anionic lipid domains: correlations with functional topography in a mammalian cell membrane. Proc Natl Acad Sci USA 77:6601–6605PubMedCrossRefGoogle Scholar
  6. Bearer EL, Friend DS (1982) Modification of anionic-lipid domains preceding membrane fusion in guinea pig sperm. J Cell Biol 92:604–615PubMedCrossRefGoogle Scholar
  7. Bendayan M, Zollinger M (1983) Ultrastructural localization of antigenic sites on osmium-fixed tissues applying the protein A-gold technique. J Histochem Cy-tochem 31:101–109CrossRefGoogle Scholar
  8. Branton D (1966) Fracture faces of frozen membranes. Proc Natl Acad Sci USA 55:1048–1055PubMedCrossRefGoogle Scholar
  9. Branton D, Moor H (1964) Fine structure in freeze-etched Allium cepa root tips. J Ultrastruct Res 11:401–411CrossRefGoogle Scholar
  10. Branton D, Cohen CM, Tyler J (1981) Interaction of cytoskeletal proteins on the erythrocyte membrane. Cell 24:24–32PubMedCrossRefGoogle Scholar
  11. Danielli JF, Davson H (1935) A contribution to the theory of permeability of thin films. J Cell Comp Physiol 5:495–508CrossRefGoogle Scholar
  12. DeMey J (1984) Colloidal gold as marker and tracer in light and electron microscopy. EMSA Bull 14:54–66Google Scholar
  13. Elias PM, Goerke J, Friend DS (1978) Freeze-fracture identification of sterol-digitonin complexes in cell and liposome membranes. J Cell Biol 78:577–596PubMedCrossRefGoogle Scholar
  14. Elias PM, Friend DS, Georke J (1979) Membrane sterol heterogeneity: Freeze-fracture detection with saponins and filipin. J Histochem Cytochem 27:1247–1260PubMedCrossRefGoogle Scholar
  15. Fawcett DW (1970) A comparative view of sperm ultrastructure. Biol Reprod 2 [Suppl]:80–127Google Scholar
  16. Fisher KA (1976) Analysis of membrane halves: cholesterol. Proc Natl Acad Sci USA 73:173–177PubMedCrossRefGoogle Scholar
  17. Fisher KA (1982 a) Monolayer freeze-fracture autoradiography: origins and directions. J Microscopy 126:1–8CrossRefGoogle Scholar
  18. Fisher KA (1982b) Monolayer freeze-fracture autoradiography: quantitative analysis of the transmembrane distribution of radioiodinated concanavalin A. J Cell Biol 93:155–163PubMedCrossRefGoogle Scholar
  19. Friend DS, Elias PM (1978) Heterogeneity of filipin-sterol complexes in the guinea pig sperm plasma membrane. J Cell Biol 79:216aGoogle Scholar
  20. Friend DS, Bearer EL (1981) β-hydroxysterol distribution as determined by freeze-fracture cytochemistry. Histochem J 13:535–546PubMedCrossRefGoogle Scholar
  21. Geoghegan WD, Ackerman GA (1977) Adsorption of horseradish peroxidase, ovomucoid and anti-immunoglobulin to colloidal gold for the indirect detection of concanavalin, wheat germ agglutinin, and goat anti-human immunoglobulin G on cell surfaces at the electron microscope level: a new method, theory, and application. J Histochem Cytochem 25:1187–1200PubMedCrossRefGoogle Scholar
  22. Goldfisher S, Kres Y, Coltoff-Schiller B, Berman J (1981) Primary fixation in osmium-potassium ferricyanide: the staining of glycogen, glycoproteins, elastine, an intranuclear reticular structure, and intercisternal trabeculae. J Histochem Cytochem 29:1105–1111CrossRefGoogle Scholar
  23. Green DL, Fleischer S (1963) The role of lipids in mitochondrial electron transfer and oxidative phosphorylation. Biochem Biophys Acta 70:554–582PubMedCrossRefGoogle Scholar
  24. Horisberger M, Rosset J (1977) Colloidal gold, a useful marker for transmission and scanning electron microscopy. J Histochem Cytochem 25:295–305PubMedCrossRefGoogle Scholar
  25. Koehler JK (1978) The mammalian sperm surface: studies with specific labeling techniques. Int Rev Cytol 54:73–108PubMedCrossRefGoogle Scholar
  26. Marchesi VT (1979) Functional proteins of the human red cell membrane. Semin Hematol 16:3–20PubMedGoogle Scholar
  27. Moor H, Miihlethaler K (1963) Fine structure in frozen-etched yeast cells. J Cell Biol 17:609PubMedCrossRefGoogle Scholar
  28. Moor H, Miihlethaler K, Waldner H, Frey-Wysshing A (1961) A new freezing-ul-tramicrotome. J Biophys Biochem Cytol 10:1–13PubMedCrossRefGoogle Scholar
  29. Nicolson GL (1974) The interaction of lectins with animal cell surfaces. Int Rev Cytol 39:89–190PubMedCrossRefGoogle Scholar
  30. Neiss WF (1984) Electron staining of the cell surface coat by osmium-low ferro-cyanide. Histochemistry 80:231–242PubMedCrossRefGoogle Scholar
  31. Peters BP, Ebism S, Goldstein IJ, Flashner M (1979) Interactions of wheat germ agglutinin with sialic acid. Biochemistry 18:5505–5511PubMedCrossRefGoogle Scholar
  32. Pinto da Silva P (1972) Translational mobility of the membrane intercalated particles of human erythrocyte ghosts: pH-dependent, reversible aggregation. J Cell Biol 53:777–787PubMedCrossRefGoogle Scholar
  33. Pinto da Silva P, Branton D (1970) Membrane splitting in freeze-etching. Covalently bound ferritin as a membrane marker. J Cell Biol 45:598–605PubMedCrossRefGoogle Scholar
  34. Pinto da Silva P, Nicolson G (1974) Freeze-etch localization of concanavalin A receptors to the membrane intercalated particles of human erythrocyte ghost membranes. Biochim Biophys Acta 363:311–319CrossRefGoogle Scholar
  35. Pinto da Silva P, Kan FWK (1984) “Label-fracture”: a method for high resolution labeling of cell surfaces. J Cell Biol 99:1156–1161PubMedCrossRefGoogle Scholar
  36. Pinto da Silva P, Torrisi MR (1982) Freeze-fracture cytochemistry: partition of gly-cophorin in freeze-fractured human erythrocyte membranes. J Cell Biol 93:463–469CrossRefGoogle Scholar
  37. Pinto da Silva P, Douglas SD, Branton D (1970) Location of A antigens on the human erythrocyte membrane. J Cell Biol 47 (2, part 2): 159aCrossRefGoogle Scholar
  38. Pinto da Silva P, Douglas SD, Branton D (1971) Localization of A antigen sites on human erythrocyte ghosts. Nature 232:194–196PubMedCrossRefGoogle Scholar
  39. Pinto da Silva P, Moss P, Fudenberg HH (1973) Anionic sites on the membrane intercalated particles of human erythrocyte ghost membranes. Freeze-etch localization. Exp Cell Res 81:127–138PubMedCrossRefGoogle Scholar
  40. Pinto da Silva P, Parkinson C, Dwyer N (1981a) Fracture-label: cytochemistry of freeze-fractured faces in the erythrocyte membrane. Proc Natl Acad Sci USA 78:343–347PubMedCrossRefGoogle Scholar
  41. Pinto da Silva P, Kachar B, Torrisi MR, Brown C, Parkinson C (1981b) Freeze-fracture cytochemistry: replicas of critical point dried cells and tissues after “fracture-label.” Science 213:230–233CrossRefGoogle Scholar
  42. Pinto da Silva P, Parkinson C, Dwyer N (1981c) Freeze-fracture cytochemistry: thin sections of cells and tissues after labeling of fracture-faces. J Histochem Cy-tochem 29:917–928CrossRefGoogle Scholar
  43. Pinto da Silva P, Torrisi MR, Kachar B (1981d) Freeze-fracture cytochemistry: Localization of wheat germ agglutinin and concanavalin A binding sites of freeze-fractured pancreatic cells. J Cell Biol 91:361–372PubMedCrossRefGoogle Scholar
  44. Robertson JD (1961) The unit membrane. In: Boyd JD, Johnson FR, Lever JD (eds) Electron microscopy in anatomy, Williams and Wilkins, Baltimore, pp 74–79Google Scholar
  45. Roth J (1983) Application of lectin-gold complexes for electron microscopic localization of glycoconjugates on thin-sections. J Histochem Cytochem 31:987–999PubMedCrossRefGoogle Scholar
  46. Rothman J, Lenard J (1977) Membrane asymmetry. Science 195:743–753PubMedCrossRefGoogle Scholar
  47. Singer SJ, Nicolson GL (1972) The fluid mosaic model of the structure of cell membranes. Science 175:720–731PubMedCrossRefGoogle Scholar
  48. Sjöstrand FS (1963) A new ultrastructural element of the membranes in mitochondria and of some cytoplasmic membrane. J Ultrastruct Res 9:340–361CrossRefGoogle Scholar
  49. Steck TL (1978) The band 3 protein of the human red cell membrane: a review. J Supramol Struct 8:311–324PubMedCrossRefGoogle Scholar
  50. Steere RL (1957) Electron microscopy of structural detail in frozen specimens. J Biophys Biochem Cytol 3:45–60PubMedCrossRefGoogle Scholar
  51. Tillack TW, Scott RE, Marchesi VT (1972) The structure of erythrocyte membranes studies by freeze-etching. II. Localization of receptors for phytohemagglutinin and influenza virus to the intramembranous particles. J Exp Med 135:1209–1227PubMedCrossRefGoogle Scholar
  52. Torrisi MR, Pinto da Silva P (1982) T lymphocyte heterogeneity: wheat germ agglutinin labeling of transmembrane glycoproteins. Proc Natl Acad Sci USA 79:5671–5674PubMedCrossRefGoogle Scholar
  53. Torrisi MR, Pinto da Silva P (1984) Compartmentalization of intracellular membrane glycocomponents is revealed by fracture-label. J Cell Biol 98:29–34PubMedCrossRefGoogle Scholar
  54. Wingvist L, Eriksson LC, Dallner G (1979) Interaction of lectins with proteins of the endoplasmic reticulum and Golgi system of rat liver. J Cell Sci 39:101–116Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1986

Authors and Affiliations

  • Pedro Pinto da Silva
  • M. L. F. Barbosa
  • A. P. Aguas

There are no affiliations available

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