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Staining Sectioned Biological Specimens for Transmission Electron Microscopy: Conventional and En Bloc Stains

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Electron Microscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1117))

Abstract

Post-staining of ultrathin sections and/or en bloc staining of specimens is necessary for differential contrast and improved resolution of cellular structures. Often specimens are fixed and stained with osmium tetroxide during fixation, but additional contrast is the result of additional heavy metal stains on the sections. The most common post-staining of sections is done on grids by aqueous uranyl acetate followed by lead citrate. When it is apparent that simple, aqueous uranium and lead post-staining is not adequate, other stains are invoked. These procedures can be as simple as en bloc staining with uranyl acetate after primary fixation and osmication. Over the years, several other treatments have been developed for use with the primary fixation or during dehydration. Tannic acid, paraphenylenediamine (PPD), and malachite green can all serve as en bloc stains and can contribute to overall improved visualization of ultrastructural details in biological specimens. Tannic acid and PPD improve membrane preservation, and malachite green is a phospholipid stain. All of these stains are compatible with aqueous fixatives and should be considered when the usual stains are not satisfactory. Marinozzi rings and microwave-assisted post-staining offer alternatives to traditional grid staining. In addition, stain precipitates on grids often can be removed by treatment with 10 % (v/v) acetic acid.

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References

  1. Gibbons IR, Bradfield JRG (1956) Experiments in staining thin-sections for electron microscopy. In: Sjöstrand FS, Rhodin J (eds) Electron microscopy proceedings of the Stockholm conference. Academic, New York, pp 121–124

    Google Scholar 

  2. Watson ML (1958) Staining of tissue sections for electron microscopy with heavy metals. J Biophys Biochem Cytol 4:475–485

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Watson ML (1958) Staining of tissue sections for electron microscopy with heavy metals: application of solutions containing lead and barium. J Biophys Biochem Cytol 4:727–729

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Reynolds ES (1963) The use of lead citrate as an opaque stain in electron microscopy. J Cell Biol 17:208–212

    Article  CAS  PubMed  Google Scholar 

  5. Hayat MA (1990) Principles and techniques of electron microscopy, vol 1. Van Nostrand Reinhold Co, New York, pp 253–264

    Google Scholar 

  6. Avery SW, Ellis EA (1978) Methods for removing uranyl acetate precipitate from ultrathin sections. Stain Technol 53:137–140

    CAS  PubMed  Google Scholar 

  7. Kuo J (1980) A simple method for removing stain precipitates from biological sections transmission electron microscopy. J Microsc (Oxford) 120:221–224

    Article  CAS  Google Scholar 

  8. Kuo J, Husca GL (1980) Removing stain precipitates from biological ultrathin sections. Micron 11:501–502

    Google Scholar 

  9. Kuo J, Husca GL, Lucas LND (1981) Forming and removing stain precipitates on ultrathin sections. Stain Technol 56:221–224

    Google Scholar 

  10. Mollenhauer HH, Morré DJ (1978) Contamination of thin sections, cause and elimination. In: Sturgess JM. (ed) Proc 9th Internat. Congr Electron Microsc II, 78–79. Toronto

    Google Scholar 

  11. Ellis EA, Anthony DW (1979) A method for removing precipitate from ultrathin sections resulting from glutaraldehyde-osmium tetroxide fixation. Stain Technol 54:282–285

    CAS  PubMed  Google Scholar 

  12. Marinozzi V (1961) Silver impregnation of ultrathin sections for electron microscopy. J Biophys Biochem Cytol 9:121–133

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. Chien K, Van de Velde RL, Heusser RC et al. (1994) A rapid phosphotungstic acid staining method on ultra-thin sections. In: Bailey GW (ed), Proc. Ann. MSA Meeting 49, 318–319

    Google Scholar 

  14. Austin RL (2001) Basic procedures for electron microscopy processing and staining in clinical laboratory using microwave oven. In: Giberson RT, Demaree RS (eds) Microwave: techniques and protocols. Humana Press, Totowa, NJ, pp 37–48

    Chapter  Google Scholar 

  15. Simionescu N, Simionescu M (1976) Galloylglucoses of low molecular weight as mordant in electron microscopy. I. Procedure, and evidence for mordanting effect. J Cell Biol 70:608–621

    Article  CAS  PubMed  Google Scholar 

  16. Simionescu N, Simionescu M (1976) Galloylglucoses of low molecular weight as mordant in electron microscopy. II. The moiety and functional groups possibly involved in the mordanting effect. J Cell Biol 70: 622–6233

    Article  CAS  PubMed  Google Scholar 

  17. Teichman RJ, Takei GH, Cummins JM (1974) Detection of fatty acids, fatty aldehydes, phospholipids, glycolipids and cholesterol on thin-layer chromatography stained with malachite green. J Chromatography 88:425–427

    Article  CAS  Google Scholar 

  18. Pourcho RG, Bernstein MH, Gould SF (1978) Malachite green: applications in electron microscopy. Stain Technol 53:29–35

    CAS  PubMed  Google Scholar 

  19. Cocchiaro JL, Kumar Y, Fischer ER et al (2008) Cytoplasmic lipid droplets are translocated into the lumen of the Chlamydia trachomatis parasitophorous vacuole. Proc Natl Acad Sci USA 105:9379–9384

    Article  CAS  PubMed  Google Scholar 

  20. Schultz RD, Ellis EA, Gumienny TL (2012) Two novel staining protocols resolve Caenorhabditis elegans structures for confocal and transmission electron microscopy. Proc Microsc Microanal 18(Suppl 2):100–101

    Article  Google Scholar 

  21. Phend KD, Rustioni A, Weinberg R (1995) An osmium free method of epon embedment that preserves both ultrastructure and antigenicity for post-embedding immunocytochemistry. J Histochem Cytochem 43:283–292

    Article  CAS  PubMed  Google Scholar 

  22. Brorson S-H, Halvorsen I, Lonning L-C et al (1999) Increased yield of immunogold labeling of epoxy sections by adding paraphenylenediamine in the tissue processing. Micron 30: 561–566

    Article  CAS  PubMed  Google Scholar 

  23. Ellis EA (2010) p-Phenylenediamine: an adjunct to and a substitute for osmium tetroxide. Microsc Today 18:48, 50

    Google Scholar 

  24. Miao R, Martinho M, Morales JG et al (2008) EPR and Mossbauer spectroscopy of intact mitochondria isolated from Yah1p-depleted Saccharomyces cerevisiae. Biochemistry 47: 9556–9568

    Article  Google Scholar 

  25. Hayat MA (1975) Positive staining for electron microscopy. Van Nostrand Reinhold Co., New York

    Google Scholar 

  26. Luft JH (1961) Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol 9:409–414

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  27. Spurr AR (1969) A low viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31–43

    Article  CAS  PubMed  Google Scholar 

  28. Mollenhauer HH (1964) Plastic embedding mixtures for use in electron microscopy. Stain Technol 39:111–114

    CAS  PubMed  Google Scholar 

  29. Daddow LYM (1983) A double stain method for enhancing contrast of ultrathin sections in electron microscopy: a multiple staining technique. J Microsc (Oxford) 129:147–153

    Article  CAS  Google Scholar 

  30. Bell M (1988) Artifacts in staining procedures. In: Crang RFE, Klomparens KL (eds) Artifacts in biological electron microscopy. Plenum Press, New York, pp 81–106

    Google Scholar 

  31. Maunsbach AB, Afzelius BA (1999) Biomedical electron microscopy: illustrated methods and interpretations. Academic, New York

    Google Scholar 

  32. Ellis EA (2006) Solutions to the problem of ERL 4221 for vinyl cyclohexene dioxide in Spurr low viscosity embedding formulations. Microsc Today 14:32–33

    CAS  Google Scholar 

  33. Mollenhauer HH (1974) Poststaining sections for electron microscopy. Stain Technol 49: 305–308

    CAS  PubMed  Google Scholar 

  34. Mollenhauer HH (1975) Poststaining sections for electron microscopy: an alternate procedure. Stain Technol 50:292

    CAS  PubMed  Google Scholar 

  35. McDowell EM, Trump BF (1976) Histologic fixatives suitable for diagnostic light and electron microscopy. Arch Pathol Lab Med 100: 405–414

    CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by the Microscopy and Imaging Center at Texas A&M University. Appreciation is extended to Robbie Schultz for C. elegans micrographs, Nema Jhurry for sections of cultured Jurkat cells, and Austin Butts for sections from kidneys of dogs.

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Authors and Affiliations

  1. Thomasville, GA, USA

    E. Ann Ellis

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  1. E. Ann Ellis
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Editors and Affiliations

  1. University of Western Australia, Crawley, Australia

    John Kuo

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Ellis, E.A. (2014). Staining Sectioned Biological Specimens for Transmission Electron Microscopy: Conventional and En Bloc Stains. In: Kuo, J. (eds) Electron Microscopy. Methods in Molecular Biology, vol 1117. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-776-1_4

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  • DOI: https://doi.org/10.1007/978-1-62703-776-1_4

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-775-4

  • Online ISBN: 978-1-62703-776-1

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