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The Histochemical Journal

, Volume 28, Issue 5, pp 375–383 | Cite as

The effects of mercaptoethanol-formaldehyde on tissue fixation and protein retention

  • B. Durgun-Yücel
  • D. Hopwood
  • A. H. Yücel
Papers

Summary

The study compared the effects of mercaptoethanol-formaldehyde and formaldehyde alone, on tissue fixation and protein retention in human and mouse tissues. Shrinkage of tissues and the penetration rate of the fixatives were assessed. The cross-linking ability of the fixatives was determined by viscometry, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and spectrophotometry, using bovine serum albumin and human haemoglobin. Tissues fixed in buffered 0.0025% mercaptoethanol-4% formaldehyde showed good nuclear and cytoplasmic detail, better than those fixed in buffered 4% formaldehyde. There was no significant difference in shrinkage. A mixture of 0.0025% mercaptoethanol-4% formaldehyde penetrated faster into adult liver than 4% formaldehyde. The mean penetration rate (±SE) or coefficient of diffusibility of 0.0025% mercaptoethanol-4% formaldehyde into adult liver was 1.32±0.01 and that of 4% formaldehyde was 1.12±0.06 (p<0.04). Both fixatives diffused more rapidly into mouse liver than into human liver. The cross-linking ability of mercaptoethanol-formaldehyde depends on the concentration of the fixative and the time of fixation. Bovine serum albumin (15%) and 0.1% mercaptoethanol alone formed a gel, whilst electrophoresis showed monomers in the supernatant. Mercaptoethanol (0.1%) also rapidly decreased the absorption at 420 nm, suggesting denaturation. It seems that mercaptoethanol increases the number of thiol groups available to form cross-links with formaldehyde. This study demonstrated that mercaptoethanol-formaldehyde fixed and cross-linked tissues better than formaldehyde at 3 h and 4 h, but not at 1 h and 2 h. The most effective concentration of mercaptoethanol for tissue fixation in 4% formaldehyde is 0.0025%.

Keywords

Formaldehyde Bovine Serum Albumin Thiol Dodecyl Sodium Dodecyl 
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.

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References

  1. Baker, J. R. &McCrae, J. M. (1966) The fine structure resulting from fixation by formaldehyde: the effects of concentration, duration and temperature.J. Royal Microsc. Soc. 85, 391–4.Google Scholar
  2. Barka, T. &Anderson, F. J. (1963)Histochemistry: Theory, Practice and Bibliography. New York: Hoeber Medical Division, Harper and Row.Google Scholar
  3. Bancroft, J. D. &Stevens, A. (1982)Theory and Practice of Histological Techniques. Edinburgh: Churchill Livingstone.Google Scholar
  4. Boon, M. E., Gerrits, P. O., Moorlag, H. E., Nieuwenhuis P. &Kok, L. P. (1988) Formaldehyde fixation and microwave irradiation.Histochem. J. 20, 313–22.PubMedCrossRefGoogle Scholar
  5. Burnett, M. G. (1982) The mechanism of the formaldehyde clock reaction. Methylene glycol dehydration.J. Chem. Ed. 59, 160–2.Google Scholar
  6. Cavallini, D., Mondovi, B., DeMarco, C. &Scioscia-Santoro, A. (1962) The mechanism of desulphhydration of cysteine.Enzymologia 24, 253–66.PubMedGoogle Scholar
  7. Chen, R. F., Scott, C. &Trepman, E. (1979) Fluorescence properties ofo-phthaldialdehyde derivatives of amino acids.Biochim. Biophys. Acta 576, 440–55.PubMedGoogle Scholar
  8. Durgun-Yücel, B., Dere, F., Yücel, A. H. &Oguz, Ö. (1992) Rapid fixation of whole organ specimens and attended problems.Acta Medica Okayama 46, 75–81.PubMedGoogle Scholar
  9. French, D. &Edsall, J. T. (1945) The reaction of formaldehyde with amino acids and proteins.Adv. Protein Chem. 2, 277–335.Google Scholar
  10. Fox, C. H., Johnson, F. B., Whiting, J. &Roller, P. P. (1985) Formaldehyde fixation.J. Histochem. Cytochem. 33, 845–53.PubMedGoogle Scholar
  11. Hays, W. L. (1988)Statistics 4th edn. New York: CBS College Publishing.Google Scholar
  12. Hopwood, D. (1970) The reactions between formaldehyde, glutaraldehyde, and osmium tetroxide, and their fixation effects on bovine serum albumin and on tissue block.Histochemie 24, 50–60.PubMedCrossRefGoogle Scholar
  13. Hopwood, D. (1982) Fixation and fixatives. InTheory and Practice of Histological Techniques (edited by J. D. Bankroft & A. Stevens) Edinburgh: Churchill Livingstone.Google Scholar
  14. Hopwood, D. (1985) Cell and tissue fixation, 1972–1982.Histochem. J. 17, 389–442.PubMedCrossRefGoogle Scholar
  15. Hopwood, D. (1989) Tissue fixation with phenol-formaldehyde for routine histopathology.Histochem. J. 21 228–34.PubMedCrossRefGoogle Scholar
  16. Keutmann, H. T. & Potts, J. T. Jr. (1969) Improved recovery of methionine after acid hydrolysis using mercaptoethanol.Anal. Biochem 29, 175–85.PubMedCrossRefGoogle Scholar
  17. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227, 680–5.PubMedCrossRefGoogle Scholar
  18. Lindeman, H. R., Merenda, P. F. &Gold, R. (1980)Introduction to Bivariate and Multivariate Analysis. New York: Scott, Foresman and Co.Google Scholar
  19. Luft, J. H. (1992) Fixation for biological ultrastructure. I. A. viscometric analysis of the interaction between glutaraldehyde and bovine serum albumin.J. Microsc. Oxford,167 247–58.Google Scholar
  20. Medawar, P. B. (1941) The rate of penetration of fixatives.J. Royal Microsc. Soc. 61, 46–57.Google Scholar
  21. Meister, A. (1965)Biochemistry of the Amino Acids. Vol. 1, 2nd edn. New York: Academic Press.Google Scholar
  22. Pearse, A. G. E. (1980)Histochemistry: Theoretical and Applied. Vol 1, 2nd edn. Edinburgh: Churchill-Livingstone.Google Scholar
  23. Roth, M. (1971) Fluorescence reaction for amino acids.Anal. Chem. 47, 880–2.Google Scholar
  24. Schyns, M. W. R. J., Huizinga, A., Vrensen, G. F. M. J., De Mul, F. M. &Greve, J. (1990) Paraformaldehyde fixation and some characteristics of lens proteins as measured by Raman microspectroscopy.Exp. Eye Res. 50, 331–3.PubMedCrossRefGoogle Scholar
  25. Von Hagens, G. (1979) Impregnation of soft biological specimens with thermosetting resins and elastomers.Anat. Rec. 194, 247–56.CrossRefGoogle Scholar
  26. Von Hagens, G. (1981) An improved method for the preservation of teaching specimens.Arch. Pathol. Lab. Med. 105 674–6.Google Scholar
  27. Walker, J. M. (1984)Methods in Molecular Biology. Proteins Clifton, NJ: Humana Press.Google Scholar

Copyright information

© Chapman & Hall 1996

Authors and Affiliations

  • B. Durgun-Yücel
    • 1
  • D. Hopwood
    • 2
  • A. H. Yücel
    • 1
  1. 1.Department of Anatomy, Faculty of MedicineÇukurova UniversityBalcali, AdanaTurkey
  2. 2.Department of PathologyNinewells Hospital and Medical SchoolDundeeUK

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