Skip to main content

Chemical and Physical Fixation of Cells and Tissues: An Overview

Abstract

Fixation is the essential first step in preserving cellular structures with the goal of keeping them as “lifelike” as possible. Two methods are normally used to “fix” biological specimens: chemical and physical fixation. Chemical fixation is the most common approach in specimen preservation. Tissues are immersed in a fixative that kills and stabilizes the cell contents. Physical fixation can include microwaving and cryopreserving samples to rapidly inactivate cellular activity. Chemical and physical fixation methods each have their own merits and limitations. This chapter provides a general overview of the chemical and physical fixation of cells and tissues. Practical issues on plant fixation methods are discussed.

Keywords

  • Chemical fixation
  • Cryofixation
  • Immunohistochemistry
  • Microwave fixation
  • Physical fixation

This is a preview of subscription content, access via your institution.

Buying options

Chapter
USD   29.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-3-319-19944-3_2
  • Chapter length: 21 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
eBook
USD   299.00
Price excludes VAT (USA)
  • ISBN: 978-3-319-19944-3
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   379.99
Price excludes VAT (USA)
Hardcover Book
USD   379.99
Price excludes VAT (USA)

References

  1. Karahara I, Kang BH (2014) High-pressure freezing and low-temperature processing of plant tissue samples for electron microscopy. Meth Mol Biol 1080:147–157

    CrossRef  Google Scholar 

  2. Ripper D, Schwarzt H, Stierhof YD (2008) Cryo-section immunolabeling of difficult to preserve specimens: advantages of cryofixation, freeze-substitution and rehydration. Biol Cell 100:109–123

    CAS  CrossRef  PubMed  Google Scholar 

  3. Jones ML (2001) To fix, to harden, to reserve-fixation: a brief history. J Histotechnol 24:155–171

    CrossRef  Google Scholar 

  4. Eltoum IE, Freenburgh J, Myers RB, Grizzle WE (2001) Introduction to the theory and practice of fixation of tissues. J Histotechnol 24:173–190

    CAS  CrossRef  Google Scholar 

  5. Eltoum I, Fredenburgh J, Myers RB, Grizzle WE (2001) Advanced concepts in fixation: 1. Effects of fixation on immunohistochemistry, reversibility of fixation and recovery of proteins, nucleic acids, and other molecules from fixed and processed tissues. 2. Developmental methods of fixation. J Histotechnol 24:201–210

    CAS  CrossRef  Google Scholar 

  6. Hayat MA (1981) Fixation for electron microscopy. Academic, New York

    Google Scholar 

  7. Hayat MA (2000) Principles and techniques of electron microscopy: biological applications, 4th edn. Cambridge University Press, New York

    Google Scholar 

  8. Glauert AM, Lewis PR (1998) Biological specimen preparation for transmission electron microscopy. Princeton University Press, Princeton

    CrossRef  Google Scholar 

  9. Bozzola JJ, Russel LD (1999) Electron microscopy, 2nd edn. Jones and Bartlett, Sudbury

    Google Scholar 

  10. Kiernan JA (1999) Histological and histochemical methods: theory and practice, 3rd edn. Butterworth-Heinemann, Oxford

    Google Scholar 

  11. Suvarna KS, Layton C, Bancroft JD (2013) Bancroft’s theory and practice of histological techniques, 7th edn. Churchill Livingstone, London

    Google Scholar 

  12. Kuo J (ed) (2014) Electron microscopy, 3rd edn. Humana Press, Totowa, New Jersey

    CrossRef  Google Scholar 

  13. Baker JR (1966) Cytological technique, 6th edn. Chapman & Hall, London

    Google Scholar 

  14. O’Brien TP, McCully ME (1981) The study of plant structure: principles and selected methods. Termarcarphi Pty. Ltd., Melbourne

    Google Scholar 

  15. Ruzin SE (1999) Plant microtechnique and microscopy. Oxford University Press, New York

    Google Scholar 

  16. McFadden GI, Melkonian M (1986) Use of a Hepes buffer for microalgal culture media and fixation for electron microscopy. Phycologia 25:551–557

    CAS  CrossRef  Google Scholar 

  17. Kumarasinghe MP, Constantine SR, Hemamali RL (1997) Methanol as an alternative fixative for cytological smears. Malays J Pathol 19:137–140

    CAS  PubMed  Google Scholar 

  18. Leong AS-Y (1994) Fixation and fixatives. In: Woods AE, Ellis RC (eds) Laboratory histopathology: a complete reference, vol 4. Churchill Livingstone, New York, pp 4.1-1-4,1–26

    Google Scholar 

  19. Fox CH, Johnson FB, Whiting J, Roller PP (1985) Formaldehyde fixation. J Histochem Cytochem 33:845–853

    CAS  CrossRef  PubMed  Google Scholar 

  20. Puchtler H, Meloan SN (1985) On the chemistry of formaldehyde fixation and its effects on immunohistochemical reactions. Histochem 82:201–204

    CAS  CrossRef  Google Scholar 

  21. Culling CFA, Allison RT, Barr WT (1985) Cellular pathology techniques, 4th edn. Butterwords, London

    Google Scholar 

  22. Sabatini DD, Bensch K, Barrnett RJ (1963) Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J Cell Biol 17:19–58

    PubMed Central  CAS  CrossRef  PubMed  Google Scholar 

  23. Hopwood D (1967) Some aspects of fixation with glutaraldehyde. J Anat 101:83–92

    PubMed Central  CAS  PubMed  Google Scholar 

  24. Kasten FH, Lala R (1975) The Feulgen reaction after glutaraldehyde fixation. Stain Technol 50:197–201

    CAS  PubMed  Google Scholar 

  25. Tagliaferro P, Tandler CJ, Ramos AJ, Saavedra JP, Brusco (1997) Immnofluorescence and glutaraldehye fixation. A new procedure based on the Schiff-quenching method. J Neurosci Meth 77:191–197

    CAS  CrossRef  Google Scholar 

  26. Karnovsky MJ (1965) A formaldehye-glutaraldehyde fixative of high osmolality for use in electron microscopy (abst). J Cell Biol 27: 137 A

    Google Scholar 

  27. Moelans CB, ter Hoeve N, van Ginkel J-W, ten Kate FJ, van Diest PJ (2011) Formaldehyde substitute fixatives. Amer J Clin Pathol 136:548–566

    CrossRef  Google Scholar 

  28. Dapson RW, Feldman AT, Wolfe D (2006) Glyoxal fixation and its relationship to immunohistochemistry. J Histotechnol 29:65–76

    CAS  Google Scholar 

  29. Salema R, Brandao I (1973) The use of PIPES buffer in the fixation of plant cells for electron microscopy. J Submicr Cytol 5:79–96

    CAS  Google Scholar 

  30. Lawton JR, Harris PJ (1977) Fixation of senescing plant tissues: sclerenchymatous fibre cells from the flowering stem of a grass. J Microscopy 112:307–318

    CrossRef  Google Scholar 

  31. Tung SH, Ye XL, Zee SY, Yeung EC (2000) The microtubular cytoskeleton during megasporogenesis in the Nun orchid, Phaius tankervillae. New Phytol 146:503–513

    CrossRef  Google Scholar 

  32. Dykstra MJ, Reuss LE (2003) Biological electron microscopy. Theory, techniques, and troubleshooting, 2nd edn. Springer, New York

    CrossRef  Google Scholar 

  33. Thornthwaite JT, Thomas RA, Leif SB, Yopp TA et al (1978) The use of electronic cell volume analysis with the AMAC II to determine the optimum glutaraldehyde fixative concentration for nucleated mammalian cells. Scan Electron Microscopy 2:1123

    Google Scholar 

  34. Parthasarathy MV (1994) Transmission electron microscopy: chemical fixation, freezing methods, and immunolocalization. In: Freeling M, Walbot V (eds) The maize handbook. Springer, New York, pp 118–134

    CrossRef  Google Scholar 

  35. Huang BQ, Strout GW, Russell SD (1993) Fertilization in Nicotiana tabacum: ultrastructural organization of propane-jet-frozen embryo sacs in vivo. Planta 191:256–264

    CrossRef  Google Scholar 

  36. Mersey B, McCully ME (1978) Monitoring of the course of fixation of plant cells. J Microscopy 114:49–76

    CrossRef  Google Scholar 

  37. Jensen WA (1962) Botanical histochemistry. Freeman, San Francisco

    Google Scholar 

  38. Gahan PB (1984) Plant histochemistry and cytochemistry—an introduction. Academic, London

    Google Scholar 

  39. Anderson G, Bancroft J (2002) Tissue processing and microtomy. In: Bancroft J, Gamble M (eds) Theory and practice of histological techniques. Churchill Livingstone, London, pp 85–107

    Google Scholar 

  40. Lancelle SA, Callahan DA, Hepler PK (1986) A method for rapid freeze fixation of plant cells. Protoplasma 131:153–165

    CrossRef  Google Scholar 

  41. Fields SD, Strout GW, Russell SD (1997) Spray-freezing freeze substitution (SFFS) of cell suspensions for improved preservation of ultrastructure. Micro Res Tech 38:315–328

    CAS  CrossRef  Google Scholar 

  42. Gilkey JC, Staehelin LA (1986) Advances in ultrarapid freezing for the preservation of cellular ultrastructure. J Elect Microsc Tech 3:177–210

    CrossRef  Google Scholar 

  43. Ding B, Turgeon R, Parthasarathy MV (1991) Routine cryofixation of plant tissue by propane jet freezing for freeze substitution. J Electron Microsc Tech 19:107–117

    CAS  CrossRef  PubMed  Google Scholar 

  44. McDonald KL, Morphew M, Verkade P, Muller-Reichert T (2007) Recent advances in high-pressure freezing. Meth Mol Biol 369:143–173

    CAS  CrossRef  Google Scholar 

  45. Monaghan P, Robertson D (1990) Freeze-substitution without aldehyde or osmium fixatives: ultrastructure and implications for immunocytochemistry. J Microscopy 158:355–363

    CAS  CrossRef  Google Scholar 

  46. Kaech A, Ziegler U (2014) High-pressure freezing: current state and future prospects. Meth Mol Biol 1117:151–172

    CAS  CrossRef  Google Scholar 

  47. King BH (2010) Electron microscopy and high-pressure freezing of Arabidopsis. Meth Cell Biol 96:259–283

    CrossRef  Google Scholar 

  48. Wilson SM, Bacic A (2012) Preparation of plant cells for transmission electron microscopy to optimize immunogold labeling of carbohydrate and protein epitopes. Nature Protoc 7:1716–1727

    CAS  CrossRef  Google Scholar 

  49. McDonald KL (2014) Out with the old and in with the new: rapid specimen preparation procedures for electron microscopy of sectioned biological material. Protoplasma 251:429–448

    CAS  CrossRef  PubMed  Google Scholar 

  50. McDonald KL, Webb RI (2011) Freeze substitution in 3 hours or less. J Microscopy 243:227–233

    CAS  CrossRef  Google Scholar 

  51. Hurbain I, Sachse M (2011) The future is cold: cryo-preparatoon method for transmission electron microscopy of cells. Biol Cell 103:405–420

    CrossRef  PubMed  Google Scholar 

  52. Mayers CP (1970) Histological fixation by microwave heating. J Clin Pathol 23:273–275

    PubMed Central  CAS  CrossRef  PubMed  Google Scholar 

  53. Login GR, Dvorak AM (1994) Methods of microwave fixation for microscopy—a review of research and clinical applications: 1970–1992. Prog Histochem Cytochem 27:1–119

    CAS  CrossRef  PubMed  Google Scholar 

  54. Giberson RT, Demaree RS Jr (eds) (2001) Microwave techniques and protocols. Humana Press, Totowa

    CrossRef  Google Scholar 

  55. Zcehmann B, Zellnig G (2009) Microwave-assisted rapid plant sample preparation for transmission electron microscopy. J Microscopy 233:258–268

    CrossRef  Google Scholar 

  56. Leria F, Marco R, Medina FJ (2004) Structural and antigenic preservation of plant samples by microwave-enhanced fixation, using dedicated hardware, minimizing heat-related effects. Micros Res Tech 65:86–100

    CrossRef  Google Scholar 

  57. Russin WA, Trivett CL (2001) Vacuum-microwave combination for processing plant tissues for electron microscopy. In: Giberson RT, Demarce RS Jr (eds) Microwave techniques and protocols. Humana Press, Totowa, pp 25–35

    CrossRef  Google Scholar 

  58. Webster P (2014) Microwave-assisted processing and embedding for transmission electron microscopy. Meth Mol Biol 1117:21–37

    CAS  CrossRef  Google Scholar 

  59. Schichnes D, Nemson JA, Ruzin SE (2001) Microwave paraffin techniques for botanical tissues. In: Giberson RT, Demarce RS Jr (eds) Microwave techniques and protocols. Humana Press, Totowa, pp 181–189

    CrossRef  Google Scholar 

  60. Schichnes D, Nemson J, Sohlberg L, Ruzin SE (1999) Microwave protocols for paraffin microtechnique and in situ localization in plants. Microsc Microanal 4:491–496

    CrossRef  Google Scholar 

  61. Zellnig G, Mostl S, Zechmann B (2013) Rapid immunohistochemical diagnosis of tobacco mosaic virus disease by microwave-assisted plant sample preparation. Microscopy 62:547–553

    PubMed Central  CAS  CrossRef  PubMed  Google Scholar 

  62. Jauneau A, Cabin-Flaman A, Morvan C, Pariot C, et al (1994) Polysaccharide distribution in the cellular junctions of immature fibre cells of flax seedlings. Histochem J 26:226–232

    CAS  CrossRef  PubMed  Google Scholar 

  63. Beneŝ K (1968) On stainability of plant cell walls with alcian blue. Biol Plant 10:334–346

    CrossRef  Google Scholar 

  64. Lawton JR (2011) The use of malachite green during fixation of plant tissues for the preservation of lipids. J Microscopy 144:201–209

    CrossRef  Google Scholar 

  65. Hughes JE, Gunning BES (1980) Glutaraldehyde-induced deposition of callose. Can J Bot 58:250–258

    CAS  CrossRef  Google Scholar 

  66. Sonobe S, Shibaoka H (1989) Cortical fine actin filaments in higher plant cells visualized by rhodamine-phalloidin after pretreatment with m-maleimidobenzoyl N-hydroxysuccinimide ester. Protoplasma 148:80–86

    CrossRef  Google Scholar 

  67. McLean W, Nakane PF (1974) Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem 22:1077–1083

    CAS  CrossRef  PubMed  Google Scholar 

  68. Swatzell LJ, Edelmann RE, Makaroff CA, Kiss JZ (1999) Integrin-like proteins are localized to plasma membrane fractions, not plastids, in Arabidopsis. Plant Cell Physiol 40:173–183

    CAS  CrossRef  PubMed  Google Scholar 

  69. Huang BQ, Sheridan WF (1994) Female gametophyte development in maize: microtubular organization and embryo sac polarity. Plant Cell 6:854–861

    CrossRef  Google Scholar 

  70. Huang BQ, Russell SD (1994) Fertilization in Nicotiana tabacum: cytoskeletal modifications in the embryo sac during synergid degeneration. A hypothesis for short-distance transport of sperm cells prior to gamete fusion. Planta 194:200–214

    CAS  CrossRef  Google Scholar 

  71. Huang BQ, Ye XL, Yeung EC, Zee SY (1998) Embryology of Cymbidium sinense: the microtubule organization of early embryos. Ann Bot 81:741–750

    CrossRef  Google Scholar 

  72. Baskin TI, Busby CH, Fowke LC, Sammut M, Gubler F (1992) Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa. Planta 187:405–413

    CAS  CrossRef  PubMed  Google Scholar 

  73. Vitha S, Baluška F, Mews M, Volkmann D (1997) Immunofluorescence detection of F-actin on low melting point wax sections from plant tissues. J Histochem Cytochem 45:89–95

    CAS  CrossRef  PubMed  Google Scholar 

  74. Griffiths G (1993) Fine structure immunocytochemistry. Springer, Berlin

    CrossRef  Google Scholar 

  75. Benhamou N, Noel S, Grenier J, Asselin A (1991) Microwave energy fixation of plant tissue: an alternative approach that provides excellent preservation of ultrastructure and antigenicity. J Electron Microsc Tech 17:81–94

    CAS  CrossRef  PubMed  Google Scholar 

  76. Heumann HG (1992) Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of plant tissue: ultrastructural preservation in seconds. Histochem 97:341–347

    CAS  CrossRef  Google Scholar 

  77. Carpentier A, Abreu S, Trichet M, Satiat-Jeunemaitre (2012) Microwaves and tea: new tools to process plant tissue for transmission electron microscopy. J Microscopy 247:94–105

    CAS  CrossRef  Google Scholar 

  78. Takahashi H, Kamakura H, Sato Y, Shiono K et al (2010) A method for obtaining high quality RNA from paraffin sections of plant tissues by laser microdissection. J Plant Res 123:807–813

    CAS  CrossRef  PubMed  Google Scholar 

  79. Belmonte MF, Kirkbride RC, Stone SL, Pelletier JM et al (2013) Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed. Proc Natl Acad Sci U S A 110:E435–E444

    PubMed Central  CAS  CrossRef  PubMed  Google Scholar 

  80. Páska C, Krisztina B, László S, Annamária T et al (2004) Effect of formalin, acetone, and RNAlater fixatives on tissue preservation and different size amplicons by real-time PCR from paraffin-embedded tissue. Diagn Molec Path 13:234–240

    CrossRef  Google Scholar 

  81. Hess MW (2003) Of plants and other pets: practical aspects of freeze-substitution and resin embedding. J Microscopy 212:44–52

    CAS  CrossRef  Google Scholar 

  82. Hess MW (2007) Cryopreparation methodology for plant cell biology. Meth Cell Biol 79:57–100

    CAS  CrossRef  Google Scholar 

  83. Austin II JR (2014) High-pressure freezing and freeze substitution of Arabidopsis for electron microscopy. Meth Mol Biol 1062:473–486

    Google Scholar 

  84. Kuo J (2014) Processing plant tissue for ultrastructural study. Meth Mol Biol 1117:39–55

    CAS  CrossRef  Google Scholar 

  85. Kiss JZ, Giddings TH Jr, Staehelin LA, Sack FD (1990) Comparison of the ultrastructure of conventionally fixed and high pressure frozen/freeze substituted root tips of Nicotiana and Arabidopsis. Protoplasma 157:64–74

    CAS  CrossRef  PubMed  Google Scholar 

  86. Stachelin LA, Giddings TH Jr, Kiss JZ, Sack FD (1990) Macromolecular differentiation of Golgi stacks in root tips of Arabidopsis and Nicotiana seedlings as visualized in high pressure frozen and freeze-substituted samples. Protoplasma 157:75–91

    CrossRef  Google Scholar 

  87. Thijssen MH, Mittempergher F, Van Aelst AC, Van Went JL (1997) Improved ultrastructural preservation of Petunia and Brassica ovules and embryo sacs by high pressure freezing and freeze substitution. Protoplasma 197:199–209

    CrossRef  Google Scholar 

  88. Kaneko Y, Walther P (1995) Comparison of ultrastructure of germinating pea leaves prepared by high-pressure freezing-freeze substitution and conventional chemical fixation. J Electron Microsc 44:104–109

    CAS  Google Scholar 

  89. Bourett TM, Czymmek KJ, Howard RJ (1999) Ultrastructure of chloroplast protuberances in rice leaves preserved by high-pressure freezing. Planta 208:472–479

    CAS  CrossRef  Google Scholar 

  90. Titford M (2001) Safety considerations in the use of fixatives. J Histotechnol 24:165–171

    CAS  CrossRef  Google Scholar 

Download references

Acknowledgments

The authors thank Professor Jeffrey L. Salisbury at the Department of Biochemistry and Molecular Biology and Microscopy and Cell Analysis Core Facility in Mayo Clinic for critical reading and editing of the manuscript.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Bing Quan Huang .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2015 Springer International Publishing Switzerland

About this chapter

Cite this chapter

Huang, B., Yeung, E. (2015). Chemical and Physical Fixation of Cells and Tissues: An Overview. In: Yeung, E., Stasolla, C., Sumner, M., Huang, B. (eds) Plant Microtechniques and Protocols. Springer, Cham. https://doi.org/10.1007/978-3-319-19944-3_2

Download citation