Histochemistry and Cell Biology

, Volume 106, Issue 1, pp 41–58 | Cite as

Improving structural integrity of cryosections for immunogold labeling

  • Willisa LiouEmail author
  • Hans J. Geuze
  • Jan W. Slot


Cryosections of aldehyde-fixed material prepared according to Tokuyasu are a good substrate for immunocytochemistry. However, structural defects occur that limit the resolution of this approach. We found that the step during which sections are thawed and transferred from the cryochamber to the supporting film on an EM grid is most critical for structural preservation. Surface tension of the transfer medium, on which sections are spread during thawing, can easily damage their structure by overstretching. By substituting a mixture of methylcellulose and sucrose for the conventional sucrose transfer medium, we were able to alleviate the problem of overstretching, thus improving greatly the structural integrity of thin cryosections. Also, material extraction from the sections after thawing causes structural damage, particularly when cross-linking is deficient. Incorporation of uranyl acetate in the transfer medium can then further help to maintain the structural integrity of the sections during the immunolabeling procedure. Excellent ultrastructure was featured in sections picked up and dried directly in methylcellulose/uranyl acetate mixtures. Such preparations can provide new insight into subcellular details and is an efficient back-up for immunolabeled sections in respect of their morphology. Cryosections from fresh frozen tissue can be preserved for immunolabeling by using transfer media that contain fixatives. This approach may have advantages if chemical fixation of tissue is thought to induce morphological artifacts or antigen redistribution.


Autophagic Vacuole Lamellar Body Direct View Fixation Strength Fresh Freeze Tissue 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Bénichou JC, Ryter A (1973) Mise ou point de la technique de coupes a congélation pour les bactéries gram+ et gram. J Microsc 17:223–232Google Scholar
  2. Bernhard W (1965) Ultramicrotomie à basse température. Annee Biol 4:5–19Google Scholar
  3. Bernhard W, Leduc EH (1967) Ultrathin frozen sections I. Methods and ultrastructural preservation. J Cell Biol 34: 757–771PubMedCrossRefGoogle Scholar
  4. Bernhard W, Nancy MT (1964) Coupes a congélation ultrafines de tissu inclus la gélatine. J Microsc 3:579–588Google Scholar
  5. Brands R, Slot JW, Geuze HJ (1983) Albumin localization in rat liver parenchymal cells. Eur J Cell Biol 32:99–107PubMedGoogle Scholar
  6. Christensen AK (1971) Frozen thin sections of fresh tissue for electron microscopy, with a description of pancreas and liver. J Cell Biol 51:772–804PubMedCrossRefGoogle Scholar
  7. Dubochet J, McDowell AW, Menge B, Schmid EN, Lickfeld KG (1983) Electronmicroscopy of frozen-hydrated bacteria. J Bacteriol 155:381–390PubMedGoogle Scholar
  8. Ebersold HR, Cordier IL, Lüthy P (1981) Bacterial mesosomes: method dependent artifacts. Arch Microbiol 130:19–21PubMedCrossRefGoogle Scholar
  9. Fengsrud M, Ross N, Berg T, Liou W, Slot JW, Seglen PO (1995) Ultrastructural and immunocytochemical characterization of autophagic vacuoles in isolated hepatocytes: effects of vinblastine and asparagine on vacuole distributions. Exp Cell Res 221:504–519PubMedCrossRefGoogle Scholar
  10. Fernández-Morán H (1952) Application of the ultrathin freezing-sectioning technique to the study of cell structures with the electron microscope. Ark Fys 4:471–491Google Scholar
  11. Genderen I van, Meer A van, Slot JW, Geuze HJ, Voorhout WF (1991) Subcellular localization of Forssman glycolipid in epithelial MDCK cells by immuno-electronmicroscopy after freeze-substitution. J Cell Biol 115:1009–1019PubMedCrossRefGoogle Scholar
  12. Geuze HJ, Kramer MF (1974) Function of coated membrane and multivesicular bodies during membrane regulation in stimulated exocrine pancreas cells. Cell Tissue Res 156:1–20PubMedCrossRefGoogle Scholar
  13. Geuze HJ, Slot JW (1980) Disproportional staining patterns of two secretory proteins in guinea pig and rat exocrine pancreatic cells. An immunoferritin and immunofluorescence study. Eur J Cell Biol 21:93–100PubMedGoogle Scholar
  14. Geuze HJ, Slot JW, Tokuyasu KT (1979) Immunocytochemical localization of amylase and chymotrypsinogen in the exocrine pancreatic cell with special attention to the Golgi complex. J Cell Biol 82:697–707.PubMedCrossRefGoogle Scholar
  15. Glickman JN, Morton PA, Slot JW, Kornfeld S, Geuze HJ (1996) The biogenesisof MHC class II compartment in human I-cell disease B lymphoblasts. J Cell Biol 132:769–785PubMedCrossRefGoogle Scholar
  16. Gordon PB, Seglen PO (1986) Use of electrical methods in the study of hepatocytic autophagy. Biomed Biochim Acta 45: 1635–1645PubMedGoogle Scholar
  17. Griffiths G (1983) Fixation for fine structure preservation and immunocytochemistry. In: Fine structure immunocytochemistry. Springer, Berlin Heidelberg New York, pp 26–89Google Scholar
  18. Griffiths g, McDowell A, Back R, Dubochet J (1984) On the preparation of cryosections for immunocytochemistry. J Ultrastruct Res 89:65–78PubMedCrossRefGoogle Scholar
  19. Haagsman HP, Gold LMG van (1991) Synthesis and assembly of lung surfactant. Annu Rev Physiol 53:441–461PubMedCrossRefGoogle Scholar
  20. Harding CV, Collins DS, Slot JW, Geuze HJ, Unanue ER (1991) Liposome-encapsulated antigens are processed in lysosomes, recycled, and presented to T cells. Cell 64:393–401PubMedCrossRefGoogle Scholar
  21. Hopkins CR, Gibson A, Shipman M, Miller K (1990) Movement of internalized ligand-receptor complexes along a continuous endosomal reticulum. nature 346:335–339PubMedCrossRefGoogle Scholar
  22. Jamicson JD, Palade GE (1967) Intracellular transport of secretory proteins in the pancreatic exocrine cell. I. Role of the peripheral elements of the Golgi complex. J Cell Biol 34:577–596CrossRefGoogle Scholar
  23. Langanger G, De Mey J (1988) Ultrathin cryosections in the plane of cell monolayers: evaluation of their potential for antibody localization studies of the cytoskeleton. J Electron Microsc Tech 8:391–399PubMedCrossRefGoogle Scholar
  24. Liou W, Chang LY, Geuze HJ, Strous GJ, Crapo JD, Slot JW (1993) Distribution of CuZn superoxide dismutase in rat liver. Free Radic Biol Med 14:201–207PubMedCrossRefGoogle Scholar
  25. Neefjes JJ, Stollorz V, Peters PJ, Geuze HJ, Ploegh HL (1990) The biosynthetic pathway of MHC class II but not of class I molecules interacts with the endocytic route. Cell 61:171–183PubMedCrossRefGoogle Scholar
  26. Orci L, Ravazzola M, Meda P, Holcomb C, Moore HP, Hicke L, Schekman R (1991) Mammalian Sec 23p homologue is restricted to the endoplasmic reticulum transitional cytoplasm. Proc Natl Acad Sci USA 88:8611–8615PubMedCrossRefGoogle Scholar
  27. Oprins A, Geuze HJ, Slot JW (1993a) Cryosubstitution dehydration of aldehyde-fixed tissue: a favorable approach to quantitative immunocytochemistry. J Histochem Cytochem 42d:497–503Google Scholar
  28. Oprins A, Duden R, Kreis TE, Geuze HJ, Slot JW (1993b) β-cop localizes mainly to the cis-Golgi side in exocrine pancreas. J Cell Biol 121:49–59PubMedCrossRefGoogle Scholar
  29. Painter RG, Tokuyasu KT, Singer SJ (1973) Immunoferrin localization of intracellular antigens: the use of ultracryotomy to obtain ultrathin sections suitable for direct immunoferritin staining. Proc Natl Acad Sci USA 70:1649–1653PubMedCrossRefGoogle Scholar
  30. Peters PJ, Neefjes JJ, Oorschot V, Ploegh HL, Geuze HJ (1991) MHC class II molecules segregate from MHC class I Molecules in the Golgi complex for transport to lysosomal compartments. Nature 349:669–676PubMedCrossRefGoogle Scholar
  31. Posthuma G, Slot JW, Geuze HJ (1987) The usefulness of the immunogold technique in quantitation of a soluble protein in ultrathin sections. J Histochem Cytochem 35:405–410PubMedGoogle Scholar
  32. Punnonen EL, Pihakaski K, Matilla K, Lounatmaa K, Hirsimäki P (1989) Intramembrane particles and fillipin labelling on the membranes of autophagic vacuoles and lysosomes in mouse liver. Cell Tissue Res 258:269–276PubMedCrossRefGoogle Scholar
  33. Rabouille C, Strous GJ, Crapo JD, Geuze HJ, Slot JW (1993) The differential degradation of two cytosolic proteins as a tool to monitor autophagy in hepatocytes by immunocytochemistry. J Cell Biol 120:897–908PubMedCrossRefGoogle Scholar
  34. Richter K, Dubochet J (1989) Gluing of vitrified specimens for cryoultramicrotomy. Experientia 45:A42Google Scholar
  35. Robinson JM, Karnovsky MJ (1991) Rapid-freezing cytochemistry: preservation of tubular lysosomes and enzyme activity. J Histochem Cytochem 39:787–792PubMedGoogle Scholar
  36. Roth J, Bendayan M, Orci L (1978) Ultrastructural localization of intracellular antigens by the use of protein A-gold complex. J Histochem Cytochem 26:1074–1081PubMedGoogle Scholar
  37. Seglen PO (1976) Preparation of isolated rat liver cells. Methods Cell Biol 13:29–83PubMedCrossRefGoogle Scholar
  38. Sesso A, de Faria FP, Iwamura ESM, Correa H (1994) A three dimensional reconstruction study of the rough ER-Golgi interface in serial thin sections of the pancreatic acinar cell of the rat. J Cell Sci 107:517–528PubMedGoogle Scholar
  39. Silva MT, Carvalho Guerra F, Magalhaes MM (1968) The fixative action of uranyl acetate in electron microscopy. Experientia 24:1074PubMedCrossRefGoogle Scholar
  40. Silva MT, Santos Mota JM, Melo JVC, Carvalho Guerra F (1971) Uranyl salts as fixatives for electron microscopy. Study of the membrane ultrastructure and phospholipid loss in bacilli. Biochim Biophys Acta 233:513–520PubMedCrossRefGoogle Scholar
  41. Slot JW, Geuze HJ (1983a) The use of protein A-colloidal gold (PAG) complexes as immunolabels in ultrathin frozen sections. In: Cuello AC (ed) Immunohistochemistry. Wiley, Chicester, pp 323–346Google Scholar
  42. Slot JW, Geuze HJ (1983b) Immunoelectron microscopic exploration of the Golgi complex. J Histochem Cytochem 31: 1049–1056PubMedGoogle Scholar
  43. Slot JW, Geuze HJ (1985) A new method of preparing gold probes for multiple-labeling cytochemistry. Eur J Cell Biol 38:87–93PubMedGoogle Scholar
  44. Slot JW, Geuze HJ, Freeman BA, Crapo JD (1986) Intracellular localization of the copper zinc and managanese superoxide dismutase in rat liver parenchymal cells. Lab Invest 55:363–369PubMedGoogle Scholar
  45. Slot JW, Geuze HJ, Gigengack S, Lienhard GE, James DE (1991) Immunolocalization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J Cell Biol 113: 123–135PubMedCrossRefGoogle Scholar
  46. Strous GJ, van Kerkhof P, Verheijen C, Rossen JWA, Liou W, Slot JW, Roelen CAM, Schwartz AL (1994) Expression of functional growth hormone receptor in a mouse L cell line infected with recombinant vaccinia virus. Exp Cell Res 211:353–359PubMedCrossRefGoogle Scholar
  47. Studer D, Michel M, Müller M (1989) High pressure freezing comes of age. Scanning Microsc Suppl 3:253–269Google Scholar
  48. Takizawa T, Robinson JM (1994) Composition of the transfer medium is crucial for high-resolution immunocytochemistry of cryosectioned human neutrophils. J Histochem Cytochem 42: 1157–1159PubMedGoogle Scholar
  49. Tokuyasu KT (1973) A technique for ultracryotomy of cell suspensions and tissues. J Cell Biol 57:551–565PubMedCrossRefGoogle Scholar
  50. Tokuyasu KT (1978) A study of positive staining of ultrathin frozen sections. J Ultrastruct Res 63:287–307PubMedCrossRefGoogle Scholar
  51. Tokuyasu KT (1986) Application of cryoultramicrotomy to immunocytochemistry. J Microsc (Oxford) 143:139–149Google Scholar
  52. Tokuyasu KT (1989) Use of poly(vinylpyrrolidone) and poly (vinyl alcohol) for cryoultramicrotomy. Histochem J 21:163–171PubMedCrossRefGoogle Scholar
  53. Tokuyasu KT, Singer SJ (1976) Improved procedures for immunoferritin labeling of ultrathin frozen sections. J Cell Biol 71: 894–906PubMedCrossRefGoogle Scholar
  54. Tsuji S (1978) Ultracryotomy of nerve-electroplaque synapses for immunocytochemistry. J Neurocytol 7:381–389PubMedCrossRefGoogle Scholar
  55. Tsuji S (1981) Attempted direct visualization of neatively stained amplified immune complex of synaptic acetylcholinesterase using cryoultramicrotomy sections. Histochemistry 72:369–375PubMedCrossRefGoogle Scholar
  56. Tsuji S, Anglade P, Daudet-Monsac M, Motelica-Heino I (1992) Cryoultramicrotomy: electrostatic transfer of dry ultrathin frozen sections on grids applied to the central nervous system. Arch Histol Cytol 55:423–428PubMedGoogle Scholar
  57. Voorhees P, Daignan E, Donselaar E van, Humphrey J, Marks MS, Peters PJ, Bonifacino JS (1995) An acidic sequence within the cytoplasmic domain of furin functions as a determinant of trans-Golgi network localization and internalization from the cell surface. EMBO J 14:4961–4975PubMedGoogle Scholar
  58. Voorhout W, Genderen I van, Meer G van, Geuze H (1991a) Preservation and immunogold localization of lipids by freeze-substitution and low temperature embedding. Scanning Microse Suppl 5:S17-S25Google Scholar
  59. Voorhout WF, Veenendaal T, Haagsman HP, Verkleij AJ, Golde LMG van, Geuze HJ (1991b) Sulfactant protein A is localized at the corner of the pulmonary tubular myelin lattice. J Histochem Cytochem 39:1331–1336PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1996

Authors and Affiliations

  1. 1.Medical School, Dept. of Cell BiologyUtrecht UniversityUtrechtThe Netherlands
  2. 2.Department of AnatomyChang Gung College of Medicine and TechnologyTaiwan, ROC

Personalised recommendations