On the chemistry of formaldehyde fixation and its effects on immunohistochemical reactions

Summary

Formalin has been recommended as an innocuous fixative for immunohistochemistry. However, several studies demonstrated impairment or blocking of antigenic activity of certain proteins. Formalin fixation was discovered accidentally by F. Blum in 1893 and its deleterious effects on various tissue structures were discussed extensively during the following decades. More recently, some authors assumed that formaldehyde bound to tissues can be largely or completely removed by washing and dehydration. According to chemical data, formaldehyde forms highly reactive methylols with uncharged amino groups. Such methylol groups yield methylene bridges with suitably spaced amides, arginine and aromatic amino acid sidechains. Only loosely bound formaldehyde is removed by washing for several hours. Residual bound formaldehyde cannot be dislodged by washing for weeks, but some formaldehyde is gradually removed when tissues are stored in water for an extended number of years. Methylene crosslinks resist treatment with high concentrations of urea, and can be broken only by drastic hydrolysis. It appears unlikely that such firmly bound formaldehyde is removed by conventional washing and dehydration procedures used in histochemistry. The superiority of methacarn, alcohol or acetone over formaldehyde fixation for immunohistochemical demonstration of prekeratin, myosin, type I and type IV collagen, laminin and fibronectin can be ascribed to the irreversible alterations of tissue proteins by formaldehyde.

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

References

  1. Altmannsberger M, Osborn M, Schauer A, Weber K (1981) Antibodies to different intermediate filament proteins. Lab Invest 45:427–434

    Google Scholar 

  2. Baker JR (1958) Principles of biological microtechnique. John Wiley & Sons, New York

    Google Scholar 

  3. Barka T, Anderson PJ (1963) Histochemistry: theory, practice and bibliography. Hoeber, New York

    Google Scholar 

  4. Barrnett RJ, Roth WD (1958) Effects of fixation on protein histochemistry. J Histochem Cytochem 6:406–415

    Google Scholar 

  5. Barton BP, Hobbs JL, Waldrop FS, Meloan SN, Puchtler H (1984) Effects of fixation on PAP reactions for prekeratin: formalin versus methacarn. Acta Anat 120:10

    Google Scholar 

  6. Blum F (1893) Der Formaldehyd als Härtungsmittel. Z wiss Mikrosk 10:314–315

    Google Scholar 

  7. Blum F (1896) Ueber Wesen und Wert der Formaldehydhärtung. Anat Anz 11:718–727

    Google Scholar 

  8. Blum F (1910) Formaldehyd. In: Ehrlich P, Krause R, Mosse M, Rosin H, Weigert K (eds) Enzyklopädie der mikroskopischen Technik, 2 Aufl, Bd 1. Urban & Schwarzenberg, Berlin Wien, pp 478–493

    Google Scholar 

  9. Blum J (1893) Formaldehyd als Conservierungsmittel. Zool Anz 9:229–231

    Google Scholar 

  10. Dell'Isola G (1895) Sul valore della formalina in istologia. Boll Acad Med Genova 10:84–94

    Google Scholar 

  11. Eccles WM (1894) Formic aldehyde as a rapid hardening reagent for animal tissues. Br Med J 1:1124

    Google Scholar 

  12. Fraenkel-Conrat H, Mecham DK (1949) The reaction of formaldehyde with proteins. VII Demonstration of intermolecular crosslinking by means of osmotic pressure measurements. J Biol Chem 177:477–486

    Google Scholar 

  13. Fraenkel-Conrat H, Olcott HS (1948a) Reaction of formaldehyde with proteins. VI Cross-linking of amino groups with phenol, imidazole or indole groups J Biol Chem 174:827–843

    Google Scholar 

  14. Fraenkel-Conrat H, Olcott HS (1948b) The reaction of formaldehyde with proteins. V Cross-linking between amino and primary amide or guanidyl groups. J Am Chem Soc 70:2673–2684

    Google Scholar 

  15. Fraenkel-Conrat H, Brandon BA, Olcott HS (1947) The reaction of formaldehyde with proteins. IV Participation of indole groups. Gramicidin. J Biol Chem 168:99–118

    Google Scholar 

  16. French D, Edsall JT (1945) The reactions of formaldehyde with amino acids and proteins. Adv Protein Chem 2:277–335

    Google Scholar 

  17. Gerngross O, Bach S (1923) Über die Verschiebung des isoelektrischen Punktes der Gelatine durch Formaldehyd. Biochem Z 143:533–541

    Google Scholar 

  18. Glenner GG (1957) The histochemical demonstration of indole derivatives by the rosindole reaction of E. Fischer. J Histochem Cytochem 5:297–304

    Google Scholar 

  19. Gustavson KH (1956) Aldehyde tanning. In: The chemistry of tanning processes. Academic Press, New York, pp 244–282

    Google Scholar 

  20. Gusterson BA, Wartburton MJ, Mitchell D, Ellison M, Neville AM, Rudland PS (1982) Distribution of myoepithelial cells and basement membrane proteins in normal breast and in benign and malignant breast diseases. Cancer Res 42:4763–4770

    Google Scholar 

  21. Gusterson BA, Wartburton MJ, Mitchell D, Kraft N, Hancock WW (1984) Invading squamous cell carcinoma can retain a basal lamina. Lab Invest 51:82–87

    Google Scholar 

  22. Hale AJ (1955) The effect of formalin on the periodic acid Schiff staining of certain types of mucus. J Histochem Cytochem 3:421–429

    Google Scholar 

  23. Holund B, Clausen PP, Clemmensen I (1981) The influence of fixation and tissue preparation on the immunohistochemical demonstration of fibronectin in human tissues. Histochemistry 72:291–299

    Google Scholar 

  24. Kaku T, Ekem JK, Lindayen C, Bailey DJ, van Nostrand AWP, Farber E (1983) Comparison of formalin-and acetone-fixation for immunohistochemical detection of carcinoembryonic antigen (CEA) and keratin. Am J Clin Pathol 80:806–815

    Google Scholar 

  25. Langeron M (1921) Précis de microscopie. Masson, Paris, pp 277–279

    Google Scholar 

  26. Lenard J, Singer SJ (1968) Alterations of the conformation of proteins in red blood cell membranes and in solutions by fixatives used in electron microscopy. J Cell Bil 37:117–121

    Google Scholar 

  27. Lubarsch O (1895) Technik. Ergeb allg Pathol pathol Anat 1:3–37

    Google Scholar 

  28. Mason P, Griffith JC (1964) Cross-linking fibrous proteins by formaldehyde. Nature 203:484–486

    Google Scholar 

  29. Meloan SN, Puchtler H (1982) Mallory bodies: lesions of hepatocytes containing proteins of the keratin-myosin-epidermin group. Histochemistry 75:445–460

    Google Scholar 

  30. Meloan SN, Barton BP, Puchtler H, Waldrop ES, Hobbs JL (1984a) Effects of formaldehyde and methacarn fixation on prekeratin. Georgia J Sci 42:31

    Google Scholar 

  31. Meloan SN, Puchtler H, Barton BP, Waldrop FS, Hobbs JL (1984b) Histochemical studies of prekeratin and epithelial myosins in various organs. Acta Anat 120:49

    Google Scholar 

  32. Nadji M, Morales AR (1983) Immunoperoxidase: Part I The technique and its pitfalls. Lab Med 14:767–771

    Google Scholar 

  33. Pearse AGE (1968) Histochemistry: theoretical and applied, 3rd edn, Vol 1. Little, Brown, Boston

    Google Scholar 

  34. Peters RH (1975) Textile chemistry, Vol III. Elsevier, Amsterdam Oxford New York

    Google Scholar 

  35. Puchtler H, Waldrop FS, Terry MS, Conner HM (1969) A combined PAS-myofibril stain for demonstration of early lesions of striated muscle. J Microsc 89:329–338

    Google Scholar 

  36. Puchtler H, Waldrop FS, Meloan SN, Terry MS, Conner HM (1970) Methacarn (methanol-Carnoy) fixation: Practical and theoretical considerations. Histochemie 21:97–116

    Google Scholar 

  37. Rath H (1972) Lehrbuch der Textilchemie, 3 Aufl. Springer, Berlin Heidelberg New York

    Google Scholar 

  38. Romeis B (1948) Mikroskopische Technik, 15 Aufl. Leibniz, München

    Google Scholar 

  39. Roulet F (1948) Methoden der pathologischen Histologie. Springer, Wien

    Google Scholar 

  40. Singer M (1952) Factors which control the staining of tissue sections with acid and basic dyes. Int Rev Cytol 1:211–255

    Google Scholar 

  41. Sternberger LA (1979) Immunocytochemistry, 2nd edn. John Wiley & Sons, New York

    Google Scholar 

  42. Vickerstaff T (1954) The physical chemistry of dyeing. Oliver and Boyd, London

    Google Scholar 

  43. Waldrop FS, Barton BP, Puchtler H, Meloan SN, Hobbs JL (1984) Studies of myosins, prekeratin and keratin in epithelial cells. Georgia J Sci 42:31–32

    Google Scholar 

  44. Waldrop FS, Puchtler H, Meloan SN (1984) Effects of collagen types and fixatives on one-step trichrome staining. J Histotechnol 7:181–184

    Google Scholar 

  45. Walker JF (1964) Formaldehyde, 3rd edn. Reinhold, New York

    Google Scholar 

  46. Warburton MJ, Mitchell D, Ormerod EJ, Rudland P (1982) Distribution of myoepithelial cells and basement membrane proteins in the resting, pregnant, lactating and involuting rat mammary gland. J Histochem Cytochem 30:667–676

    Google Scholar 

  47. Zeiger K (1930) Der Einfluss von Fixationsmitteln auf die Färbbarkeit histologischer Elemente. Z Zellforsch 10:481–510

    Google Scholar 

  48. Zeiger K (1938) Physikochemische Grundlage der histologischen Methodik. Theodor Steinkopff, Dresden Leipzig

    Google Scholar 

Download references

Author information

Affiliations

Authors

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Puchtler, H., Meloan, S.N. On the chemistry of formaldehyde fixation and its effects on immunohistochemical reactions. Histochemistry 82, 201–204 (1985). https://doi.org/10.1007/BF00501395

Download citation

Keywords

  • Formaldehyde
  • Laminin
  • Aromatic Amino Acid
  • Methylol
  • Tissue Protein