, Volume 82, Issue 3, pp 201–204 | Cite as

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

  • H. Puchtler
  • S. N. Meloan


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.


Formaldehyde Laminin Aromatic Amino Acid Methylol Tissue Protein 
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  1. Altmannsberger M, Osborn M, Schauer A, Weber K (1981) Antibodies to different intermediate filament proteins. Lab Invest 45:427–434Google Scholar
  2. Baker JR (1958) Principles of biological microtechnique. John Wiley & Sons, New YorkGoogle Scholar
  3. Barka T, Anderson PJ (1963) Histochemistry: theory, practice and bibliography. Hoeber, New YorkGoogle Scholar
  4. Barrnett RJ, Roth WD (1958) Effects of fixation on protein histochemistry. J Histochem Cytochem 6:406–415Google 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 120:10Google Scholar
  6. Blum F (1893) Der Formaldehyd als Härtungsmittel. Z wiss Mikrosk 10:314–315Google Scholar
  7. Blum F (1896) Ueber Wesen und Wert der Formaldehydhärtung. Anat Anz 11:718–727Google 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 478–493Google Scholar
  9. Blum J (1893) Formaldehyd als Conservierungsmittel. Zool Anz 9:229–231Google Scholar
  10. Dell'Isola G (1895) Sul valore della formalina in istologia. Boll Acad Med Genova 10:84–94Google Scholar
  11. Eccles WM (1894) Formic aldehyde as a rapid hardening reagent for animal tissues. Br Med J 1:1124Google 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 177:477–486Google 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 174:827–843Google 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 70:2673–2684Google 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 168:99–118Google Scholar
  16. French D, Edsall JT (1945) The reactions of formaldehyde with amino acids and proteins. Adv Protein Chem 2:277–335Google Scholar
  17. Gerngross O, Bach S (1923) Über die Verschiebung des isoelektrischen Punktes der Gelatine durch Formaldehyd. Biochem Z 143:533–541Google Scholar
  18. Glenner GG (1957) The histochemical demonstration of indole derivatives by the rosindole reaction of E. Fischer. J Histochem Cytochem 5:297–304Google Scholar
  19. Gustavson KH (1956) Aldehyde tanning. In: The chemistry of tanning processes. Academic Press, New York, pp 244–282Google 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 42:4763–4770Google Scholar
  21. Gusterson BA, Wartburton MJ, Mitchell D, Kraft N, Hancock WW (1984) Invading squamous cell carcinoma can retain a basal lamina. Lab Invest 51:82–87Google Scholar
  22. Hale AJ (1955) The effect of formalin on the periodic acid Schiff staining of certain types of mucus. J Histochem Cytochem 3:421–429Google 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 72:291–299Google 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 80:806–815Google Scholar
  25. Langeron M (1921) Précis de microscopie. Masson, Paris, pp 277–279Google 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 37:117–121Google Scholar
  27. Lubarsch O (1895) Technik. Ergeb allg Pathol pathol Anat 1:3–37Google Scholar
  28. Mason P, Griffith JC (1964) Cross-linking fibrous proteins by formaldehyde. Nature 203:484–486Google Scholar
  29. Meloan SN, Puchtler H (1982) Mallory bodies: lesions of hepatocytes containing proteins of the keratin-myosin-epidermin group. Histochemistry 75:445–460Google Scholar
  30. Meloan SN, Barton BP, Puchtler H, Waldrop ES, Hobbs JL (1984a) Effects of formaldehyde and methacarn fixation on prekeratin. Georgia J Sci 42:31Google 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 120:49Google Scholar
  32. Nadji M, Morales AR (1983) Immunoperoxidase: Part I The technique and its pitfalls. Lab Med 14:767–771Google Scholar
  33. Pearse AGE (1968) Histochemistry: theoretical and applied, 3rd edn, Vol 1. Little, Brown, BostonGoogle Scholar
  34. Peters RH (1975) Textile chemistry, Vol III. Elsevier, Amsterdam Oxford New YorkGoogle 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 89:329–338Google Scholar
  36. Puchtler H, Waldrop FS, Meloan SN, Terry MS, Conner HM (1970) Methacarn (methanol-Carnoy) fixation: Practical and theoretical considerations. Histochemie 21:97–116Google Scholar
  37. Rath H (1972) Lehrbuch der Textilchemie, 3 Aufl. Springer, Berlin Heidelberg New YorkGoogle Scholar
  38. Romeis B (1948) Mikroskopische Technik, 15 Aufl. Leibniz, MünchenGoogle Scholar
  39. Roulet F (1948) Methoden der pathologischen Histologie. Springer, WienGoogle Scholar
  40. Singer M (1952) Factors which control the staining of tissue sections with acid and basic dyes. Int Rev Cytol 1:211–255Google Scholar
  41. Sternberger LA (1979) Immunocytochemistry, 2nd edn. John Wiley & Sons, New YorkGoogle Scholar
  42. Vickerstaff T (1954) The physical chemistry of dyeing. Oliver and Boyd, LondonGoogle 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 42:31–32Google Scholar
  44. Waldrop FS, Puchtler H, Meloan SN (1984) Effects of collagen types and fixatives on one-step trichrome staining. J Histotechnol 7:181–184Google Scholar
  45. Walker JF (1964) Formaldehyde, 3rd edn. Reinhold, New YorkGoogle 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 30:667–676Google Scholar
  47. Zeiger K (1930) Der Einfluss von Fixationsmitteln auf die Färbbarkeit histologischer Elemente. Z Zellforsch 10:481–510Google Scholar
  48. Zeiger K (1938) Physikochemische Grundlage der histologischen Methodik. Theodor Steinkopff, Dresden LeipzigGoogle Scholar

Copyright information

© Springer-Verlag 1985

Authors and Affiliations

  • H. Puchtler
    • 1
  • S. N. Meloan
    • 1
  1. 1.Department of PathologyMedical College of GeorgiaAugustaUSA

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