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MeSH Review

Freeze Substitution

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Disease relevance of Freeze Substitution


High impact information on Freeze Substitution

  • The glutamate transporters GLT-1 and GLAST were studied by immunogold labeling on ultrathin sections of rat brain tissue embedded in acrylic resins at low temperature after freeze substitution [4].
  • Labeling with a well defined mAb and protein A-gold after freeze-substitution and low temperature embedding in Lowicryl HM20 of aldehyde-fixed and cryoprotected cells, resulted in high levels of specific labeling and excellent retention of cellular ultrastructure compared to ultra-thin cryosections [5].
  • The extracellular matrix of epiphyseal cartilage tissue was preserved in a state believed to resemble closely that of native tissue following processing by high pressure freezing, freeze substitution, and low temperature embedding (HPF/FS) [6].
  • To address concerns about using chemical fixation to study the membrane-bound transport of cationized ferritin, protoplasts were fixed both by conventional glutaraldehyde fixation and by rapid freezing in a Balzers high-pressure freezing apparatus (followed by freeze substitution) [7].
  • Rapid freezing followed by freeze-substitution has been used to study the ultrastructure of the myosin filaments of live and demembranated frog sartorius muscle in the states of relaxation and rigor [8].

Biological context of Freeze Substitution


Anatomical context of Freeze Substitution


Associations of Freeze Substitution with chemical compounds

  • We developed a new procedure, which entails application of high-pressure freezing followed by freeze-substitution with acetone containing uranyl acetate, followed by low temperature embedding in HM20 [15].
  • Portions of muscle containing the activated spindles were quick-frozen, fixed in absolute ethanol during freeze-substitution, and then embedded in paraffin wax [16].
  • To address this concern we examined activated frog nodes processed by three different methods: (i) rapid freezing/freeze-substitution, (ii) direct osmication, and (iii) primary fixation in a mixture of aldehydes and osmium [17].
  • Ultrarapid freezing, followed by freeze-fracture and freeze substitution, has confirmed aspects of the proposed exocytotic sequence, suggesting that this release is not due to the application of tannic acid, and these techniques also produced further evidence for the existence of the granule-core coat [18].
  • Olfactory bulbs were examined using freeze substitution, silver trapping of chloride and intensification techniques at light and electron microscopic level [19].

Gene context of Freeze Substitution

  • Immuno-gold-labelling of CUT-1, CUT-2 and cuticlin epitopes in Caenorhabditis elegans and Heterorhabditis sp. processed by high pressure freezing and freeze-substitution [20].
  • After vitrification, freeze-substitution and embedding of the platelets, annexin V was located on ultra thin sections, as detected by an anti-annexin V antibody and gold labelled protein A. Upon activation, the platelets show two different forms; irregular platelets with unchanged cytoplasm and round cells with apparently diluted cytoplasm [21].
  • We have compared the measurement of sodium, potassium, and chloride contents in a salt tolerant unicellular alga, Dunaliella parva, following either freeze-substitution (using two different resins) or molecular distillation drying [22].
  • METHODS: Thin slices of the parallel, tightly packed capillary bed of the eel rete mirabile were cryofixed and prepared for conventional TEM by freeze-substitution [23].
  • In this study, we have re-examined the AA amyloid fibrils with advanced methods of cryofixation and freeze substitution which are known to retain ultrastructural detail as close as possible to the living state [24].

Analytical, diagnostic and therapeutic context of Freeze Substitution


  1. Distribution of fluid in bronchovascular bundles with permeability lung edema induced by alpha-naphthylthiourea in dogs. A morphometric study. Michel, R.P., Smith, T.T., Poulsen, R.S. Lab. Invest. (1984) [Pubmed]
  2. Immunocytochemical detection of oncomodulin in tumor tissue. Brewer, L.M., Durkin, J.P., MacManus, J.P. J. Histochem. Cytochem. (1984) [Pubmed]
  3. Cryofixation combined with physical dehydration for quantitative immunoelectron cytochemistry. Eneström, S., Kniola, B. Biotechnic & histochemistry : official publication of the Biological Stain Commission. (1994) [Pubmed]
  4. Glutamate transporters in glial plasma membranes: highly differentiated localizations revealed by quantitative ultrastructural immunocytochemistry. Chaudhry, F.A., Lehre, K.P., van Lookeren Campagne, M., Ottersen, O.P., Danbolt, N.C., Storm-Mathisen, J. Neuron (1995) [Pubmed]
  5. Subcellular localization of Forssman glycolipid in epithelial MDCK cells by immuno-electronmicroscopy after freeze-substitution. van Genderen, I.L., van Meer, G., Slot, J.W., Geuze, H.J., Voorhout, W.F. J. Cell Biol. (1991) [Pubmed]
  6. Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. II. Intercellular matrix ultrastructure - preservation of proteoglycans in their native state. Hunziker, E.B., Schenk, R.K. J. Cell Biol. (1984) [Pubmed]
  7. Ultrastructure of the endocytotic pathway in glutaraldehyde-fixed and high-pressure frozen/freeze-substituted protoplasts of white spruce (Picea glauca). Galway, M.E., Rennie, P.J., Fowke, L.C. J. Cell. Sci. (1993) [Pubmed]
  8. Structure of the myosin filaments of relaxed and rigor vertebrate striated muscle studied by rapid freezing electron microscopy. Craig, R., Alamo, L., Padrón, R. J. Mol. Biol. (1992) [Pubmed]
  9. Aldehyde fixation causes membrane vesiculation during platelet exocytosis: a freeze-substitution study. Morgenstern, E. Scanning Microsc. Suppl. (1991) [Pubmed]
  10. Regional organization of astrocytic membranes in cerebellar cortex. Landis, D.M., Reese, T.S. Neuroscience (1982) [Pubmed]
  11. Rapid-freezing cytochemistry: preservation of tubular lysosomes and enzyme activity. Robinson, J.M., Karnovsky, M.J. J. Histochem. Cytochem. (1991) [Pubmed]
  12. Comparison of the ability of freeze etch and freeze substitution to preserve actin filament structure. Bridgman, P.C., Lewis, A.K., Victor, J.C. Microsc. Res. Tech. (1993) [Pubmed]
  13. Fluid dynamics of the excretory flow of zymogenic and mucin contents in rat gastric gland processed by high-pressure freezing/freeze substitution. Sawaguchi, A., Ishihara, K., Kawano Ji, J., Oinuma, T., Hotta, K., Suganuma, T. J. Histochem. Cytochem. (2002) [Pubmed]
  14. Morphogenesis of the photoreceptor outer segment during postnatal development in the mouse (BALB/c) retina. Obata, S., Usukura, J. Cell Tissue Res. (1992) [Pubmed]
  15. High-pressure freezing provides new information on human epidermis: simultaneous protein antigen and lamellar lipid structure preservation. Study on human epidermis by cryoimmobilization. Pfeiffer, S., Vielhaber, G., Vietzke, J.P., Wittern, K.P., Hintze, U., Wepf, R. J. Invest. Dermatol. (2000) [Pubmed]
  16. Distribution of fusimotor axons to intrafusal muscle fibres in cat tenuissimus spindles as determined by the glycogen-depletion method. Barker, D., Emonet-Dénand, F., Harker, D.W., Jami, L., Laporte, Y. J. Physiol. (Lond.) (1976) [Pubmed]
  17. Activity associated ultrastructural changes in peripheral nodes of Ranvier are independent of fixation. Wurtz, C.C., Ellisman, M.H. Exp. Neurol. (1988) [Pubmed]
  18. Arrested exocytosis of atrial secretory granules. Newman, T.M., Severs, N.J. J. Mol. Cell. Cardiol. (1990) [Pubmed]
  19. Chloride is preferentially accumulated in a subpopulation of dendrites and periglomerular cells of the main olfactory bulb in adult rats. Siklós, L., Rickmann, M., Joó, F., Freeman, W.J., Wolff, J.R. Neuroscience (1995) [Pubmed]
  20. Immuno-gold-labelling of CUT-1, CUT-2 and cuticlin epitopes in Caenorhabditis elegans and Heterorhabditis sp. processed by high pressure freezing and freeze-substitution. Favre, R., Hermann, R., Cermola, M., Hohenberg, H., Müller, M., Bazzicalupo, P. J. Submicrosc. Cytol. Pathol. (1995) [Pubmed]
  21. Ultrastructural detection of surface exposed phosphatidylserine on activated blood platelets. Stuart, M.C., Bevers, E.M., Comfurius, P., Zwaal, R.F., Reutelingsperger, C.P., Frederik, P.M. Thromb. Haemost. (1995) [Pubmed]
  22. Use of freeze-substitution and molecular distillation drying in the preparation of Dunaliella parva for ion localization studies by X-ray microanalysis. Hajibagheri, M.A., Flowers, T.J. Microsc. Res. Tech. (1993) [Pubmed]
  23. Linear gap and tight junctional assemblies between capillary endothelial cells in the eel rete mirabile. Wagner, R., Kachar, B. Anat. Rec. (1995) [Pubmed]
  24. AA protein in experimental murine AA amyloid fibrils: a high resolution ultrastructural and immunohistochemical study comparing aldehyde-fixed and cryofixed tissues. Inoue, S., Kuroiwa, M., Kisilevsky, R. Amyloid (2002) [Pubmed]
  25. Ultrastructural examination of the lipopolysaccharides of Pseudomonas aeruginosa strains and their isogenic rough mutants by freeze-substitution. Lam, J.S., Graham, L.L., Lightfoot, J., Dasgupta, T., Beveridge, T.J. J. Bacteriol. (1992) [Pubmed]
  26. Freeze-substitution as a preparative technique for immunoelectronmicroscopy: evaluation by atomic force microscopy. Moreira, J.E., Reese, T.S., Kachar, B. Microsc. Res. Tech. (1996) [Pubmed]
  27. Ciliated and microvillous structures of rat olfactory and nasal respiratory epithelia. A study using ultra-rapid cryo-fixation followed by freeze-substitution or freeze-etching. Menco, B.P. Cell Tissue Res. (1984) [Pubmed]
  28. Immunoelectron microscopy of acetylcholine receptors and 43 KD protein after rapid freezing, freeze-substitution, and low-temperature embedding in lowicryl K11M. Phillips, G.W., Bridgman, P.C. J. Histochem. Cytochem. (1991) [Pubmed]
  29. Chloride distribution in the CA1 region of newborn and adult hippocampus by light microscopic histochemistry. Barna, B., Kuhnt, U., Siklós, L. Histochem. Cell Biol. (2001) [Pubmed]
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