Immunostaining Methods for Frozen and Paraffin Sections

  • B. L. Mepham
  • K. J. M. Britten
Part of the Immunology and Medicine Series book series (IMME, volume 15)


The science of immunocytochemistry has developed from the simple ‘direct’ immunofluorescence test introduced by Albert Coons and colleagues1. The ‘direct’ method (Figure 1a) in which the antibody is conjugated to a fluoro-chrome or enzyme label has been progressively replaced by the ‘indirect’ method2. This method does not require the primary antibody to be labelled, as a second labelled antibody is used to detect the primary antibody (Figure 1b). Until the introduction of peroxidase labels34 the techniques were limited by the fluorescent label to frozen sections. Despite publication of a method applying the immunofluorescence technique to specially prepared paraffin embedded tissues5 considerable disadvantages still remain.


Tris Buffer Saline Paraffin Section Lymphoproliferative Disease Background Staining Unlabelled Antibody 
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  1. 1.
    Coons, A. H., Creech, H. J., Jones, R. N. and Berliner, E. (1942). The demonstration of pneumococcal antigen of tissues by the use of fluorescent antibody. J. Immunol., 45, 159–70Google Scholar
  2. 2.
    Weller, T. H. and Coons, A. H. (1954). Fluorescent antibody studies with agents of Varicella and Herpes Zoster propagated in vitro. Proceedings of the Society for Experimental Biology and Medicine, 86, 789–94Google Scholar
  3. 3.
    Sternberger, L. A. (1969). Some new developments in immunocytochemistry. Mikroskopie, 25, 346–61PubMedGoogle Scholar
  4. 4.
    Mason, T. E., Phifer, R. F., Spicer, S. S., Swallow, R. A. and Dreskin, R. B. (1969). An immunoglobulin-enzyme bridge method for localising tissue antigens. J. Histochem. Cytochem., 17, 563–9PubMedCrossRefGoogle Scholar
  5. 5.
    Sainte-Marie, G. (1962). A paraffin embedding technique for studies employing immunofluorescence. J. Histochem. Cytochem., 10, 250–6CrossRefGoogle Scholar
  6. 6.
    Sternberger, L. A., Hardy, P. H. Jr., Cuculis, J. J. and Meyer, H. G. (1970). The unlabelled antibody enzyme method of immunohistochemistry. J. Histochem. Cytochem., 18, 315–33PubMedCrossRefGoogle Scholar
  7. 7.
    Taylor, C. R. and Burns, J. (1974). The demonstration of plasma cells and other immunoglobulin-containing cells in formalin fixed, paraffin embedded tissues using peroxidase-labelled antibody. J. Clin. PathoL, 27, 14–20PubMedCrossRefGoogle Scholar
  8. 8.
    Denk, H., Radaszkiewicz, T. and Weirich, E. (1977). Pronase pre-treatment of tissue sections enhances sensitivity of the unlabelled antibody-enzyme (PAP) technique. J. Immunol. Meth., 15, 163–7CrossRefGoogle Scholar
  9. 9.
    Curran, R. C. and Gregory, J. (1977). The unmasking of antigens in paraffin sections of tissue by trypsin. Experimentia, 33, 1400–1CrossRefGoogle Scholar
  10. 10.
    Petrali, J. P., Hinton, D. M., Moriarty, G. C. and Sternberger, L. A. (1974). The unlabelled antibody enzyme method of immunocytochemistry. Quantitative comparison of sensitivities with and without peroxidase-antiperoxidase complex. (PAP). J. Histochem. Cytochem., 22, 782–801PubMedCrossRefGoogle Scholar
  11. 11.
    Burns, J., Hambridge, M. and Taylor, C. R. (1974). Intracellular immunoglobulins: A comparative study on three standard tissue processing methods using horseradish peroxidase and fluorochrome conjugates. J. Clin. Pathol., 27, 548–57PubMedCrossRefGoogle Scholar
  12. 12.
    Burns, J. (1975). Background staining and sensitivity of the unlabelled antibody (PAP) method. Comparison with the peroxidase labelled antibody sandwich method using formalin fixed, paraffin embedded material. Histochemistry, 43, 291–4PubMedCrossRefGoogle Scholar
  13. 13.
    Cordell, J. L., Falini, B., Erber, W. N., Ghosh, A. K., Abdulaziz, Z., MacDonald, S., Pulford, K. A. F., Stein, H. and Mason, D. Y. (1984). Immunoenzymatic labelling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal alkaline phosphatase (APAAP complexes). J. Histochem. Cytochem., 32, 219–29PubMedCrossRefGoogle Scholar
  14. 14.
    Suffin, S. C., Muck, K. B., Young, J. C., Lewin, K. and Parten, D. D. (1979). Improvement of the glucose oxidase immunoenzyme technique. Am. J. Clin. PathoL, 71, 492–6PubMedGoogle Scholar
  15. 15.
    Clark, C. A., Downs, E. C., Primus, F. J. (1982). An unlabelled antibody method using glucose oxidase-antiglucose oxidase complexes (GAG): A sensitive alternative to immunoperoxidase for the detection of tissue antigens. J. Histochem. Cytochem., 30, 27–34PubMedCrossRefGoogle Scholar
  16. 16.
    Bondi, I., Chieregatti, G., Eusibi, V., Fulcheri, E. and Bussolati, G. (1982). The use of beta-galactosidase as a tracer in immunocytochemistry. Histochemistry, 76, 153–8PubMedCrossRefGoogle Scholar
  17. 17.
    Sternberger, L. A. (1986). Immunocytochemistry. 3rd Edn. ( New York: John Wiley & Sons )Google Scholar
  18. 18.
    Ordronneau, P., Lindstrom, P. B-M. and Petrusz, P. (1981). Four unlabelled antibody bridge techniques: A comparison. J. Histochem. Cytochem., 29, 1397–404PubMedCrossRefGoogle Scholar
  19. 19.
    Gallyas, F., Gores, T. and Merchenthaler, I. (1982). High grade intensification of the end-product of the diaminobenzidine reaction for peroxidase histochemistry. J. Histochem. Cytochem., 30, 183–4PubMedCrossRefGoogle Scholar
  20. 20.
    Borowitz, M. J., Croker, B. P. and Burchette, J. (1982). Immunocytochemical detection of lymphocyte surface antigens in fixed tissue sections. J. Histochem. Cytochem., 30, 171–4PubMedCrossRefGoogle Scholar
  21. 21.
    Hsu, S. M. and Soban, E. (1982). Color modification of diaminobenzidine (DAB) precipitation metallic ions and its application for double immunohistochemistry. J. Histochem. Cytochem., 30, 1079–82PubMedCrossRefGoogle Scholar
  22. 22.
    Adams, J. C. (1981). Heavy metal intensification of DAB-based HRP reaction product. J. Histochem. Cytochem., 29, 775PubMedCrossRefGoogle Scholar
  23. 23.
    Hsu, S. M., Raine, L. and Fanger, H. (1981). Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabelled antibody (PAP) procedures. J. Histochem. Cytochem., 29, 577–80PubMedCrossRefGoogle Scholar
  24. 24.
    Hoffman, K., Titus, G., Montibeller, J. A. and Finn, F. M. (1982). Avidin binding of carboxyl-substituted biotin and analogues. Biochemistry, 21, 978–84CrossRefGoogle Scholar
  25. 25.
    Bonnard, C., Papermaster, D. S. and Kraehenbuhl, J. P. (1984). The streptavidin-biotin bridge technique: Application in light and electron microscope immunocytochemistry. In Polak, J. M. and Varndell, I. M. (eds), Immunolabelling for Electron Microscopy, pp. 95–111. ( Amsterdam: Elsevier Scientific )Google Scholar
  26. 26.
    Faulk, W. P. and Taylor, G. M. (1971). An immunocolloid method for the electron microscope. Immunochemistry, 8, 1081–3PubMedCrossRefGoogle Scholar
  27. 27.
    Danscher, G. (1981). Localisation of gold in biological tissue. A photochemical method for light and electron microscopy. Histochemistry, 71, 81–8PubMedCrossRefGoogle Scholar
  28. 28.
    Holgate, C. S., Jackson, P., Cowen, P. N. and Bird, C. C. (1983). Immunogold-silver staining: A new method of immunostaining with enhanced sensitivity. J. Histochem. Cytochem., 31, 938–44PubMedCrossRefGoogle Scholar
  29. 29.
    Springall, D. R., Hacker, G. W., Grimelius, L. and Polak, J. (1984). The potential of the immunogold-silver staining method for paraffin sections. Histochemistry, 81, 603–8PubMedCrossRefGoogle Scholar
  30. 30.
    Burns, J. (1975). An appraisal of immunocytochemical methods in routine histology. Proc. R. Microsc. Soc. 10, 97Google Scholar
  31. 31.
    Straus, W. (1976). Use of peroxidase inhibitors for immunoperoxidase procedures. In Felman, (ed.) International Symposium on Immunoenzymatic Techniques. INSERM Symposium No. 2, pp. 117–124Google Scholar
  32. 32.
    Heyderman, E. (1979). Immunopathology technique in histopathology: Applications, methods and controls. J. Clin. Pathol., 2, 971–8CrossRefGoogle Scholar
  33. 33.
    Streefkerk, J. G. (1972). Inhibition of erythrocyte pseudoperoxidase activity by treatment with hydrogen peroxide following methanol. J. Histochem. Cytochem., 20, 829–31PubMedCrossRefGoogle Scholar
  34. 34.
    Brooks, D. A., Zola, H., McNamara, P. J., Bradley, J., Bradstock, K. F., Hancock, W. W. and Atkins, R. C. (1983). Membrane antigens of human cells of the monocyte/ macrophage lineage studied with monoclonal antibodies. Pathology, 15, 45–52Google Scholar
  35. 35.
    Ponder, B. A. and Wilkinson, M. M. (1981). Inhibition of endogenous tissue alkaline phosphatase with use of alkaline phosphatase conjugates in immunocytochemistry. J. Histochem. Cytochem., 29, 981–4PubMedCrossRefGoogle Scholar
  36. 36.
    Graham, R. C. Jr. and Karnovsky, M. J. (1966). The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem., 14, 291–302PubMedCrossRefGoogle Scholar
  37. 37.
    Graham, R. C. Jr., Ludholm, U. and Karnovsky, M. J. (1965). Cytochemical demonstration of peroxidase activity with 3-amino-9-ethylcarbozole. J. Histochem. Cytochem., 13, 150–2PubMedCrossRefGoogle Scholar
  38. 38.
    Hanker, J. S., Yates, P. E., Metz, C. B. and Rustioni, A. (1977). A new specific, sensitive and non-carcinogenic reagent for the demonstration of horseradish peroxidase. Histochem. J., 9, 789–92PubMedCrossRefGoogle Scholar
  39. 39.
    Bancroft, J. D. and Stevens, A. (1982). Theory and Practice of Histological Techniques. 2nd Edn, pp. 381–384. ( London: Churchill Livingstone )Google Scholar
  40. 40.
    Mason, D. Y., Sammons, R. E. (1978). Alkaline phosphatase and peroxidase for double immunoenzymatic labelling of cellular constituents. J. Clin. Pathol., 31, 454–62PubMedCrossRefGoogle Scholar
  41. 41.
    Isaacson, P. G. and Wright, D. H. (1986). In Polak, J. M. and van Noorden, S. (eds) Immunocytochemistry Modern Methods and Applications. 2nd Edn. (Bristol: Wright)Google Scholar
  42. 42.
    Lillie, R. D. (1965). Histopathologic Technique and Practical Histochemistry. ( London: MacGraw-Hill )Google Scholar
  43. 43.
    Bancroft, J. D. (1967). An Introduction to Histochemical Technique. Chapter 5, pp. 54–59. ( London: Butterworths )Google Scholar
  44. 44.
    Garvin, A. J., Spicer, S. S., Parmley, R. T. and Munster, A. M. (1974). Immunohistochemical demonstration of IgG in Reed-Sternbert and other cells in Hodgkin’s disease. J. Exp. Med., 139, 1077–83PubMedCrossRefGoogle Scholar
  45. 45.
    Nakane, P. K. (1975). Recent progress in the peroxidase labelled antibody method. Ann. NY Acad. Sci., 254, 203–11PubMedCrossRefGoogle Scholar
  46. 46.
    Curran, R. C. and Gregory, J. (1980). Effects of fixation and processing on immunohistochemical demonstration of immunoglobulin in paraffin sections of tonsil and bone marrow. J. Clin. Pathol., 33, 1047–57PubMedCrossRefGoogle Scholar
  47. 47.
    Mepham, B. L. (1982). Influence of fixatives on the immunoreactivity of paraffin sections. Histochem. J., 14, 731–7PubMedCrossRefGoogle Scholar
  48. 48.
    Epenetos, A. A., Bobrow, L. G., Adams, T. E., Collins, C. M., Isaacson, P. G. and Bodmer, W. F. (1985). A monoclonal antibody that detects HLA-D region antigen in routinely fixed, wax embedded sections of normal and neoplastic lymphoid tissues. J. Clin. Pathol., 38, 12–17PubMedCrossRefGoogle Scholar
  49. 49.
    Mitchell, D., Salih Ibrahim and Gusterson, B. A. (1985). Improved immunohistochemical localization of tissue antigens using modified methacarn fixation. J. Histochem. Cytochem., 33, 491–5PubMedCrossRefGoogle Scholar
  50. 50.
    Puchtler, H., Sweat Waldrop, F., Meloan, S. N., Terry, M. S. and Conner, H. M. (1970). Methacarn (Methanol-Carnoy) fixation. Practical and theoretical considerations. Histochemie, 21, 97–116PubMedCrossRefGoogle Scholar
  51. 51.
    Baker, J. R. (1963). In Principles of Biological Microtechnique. (London: Methuen)Google Scholar
  52. 52.
    van Noorden, S. (1986). Tissue preparation and immunostaining techniques for light microscopy. In Polak, J. M. and van Noorden, S. (eds) Immunohistochemistryy: Modern Methods and Applications. 2nd Edn. ( Bristol: Wright )Google Scholar
  53. 53.
    Reading, M. (1977). A digestion technique for reduction of background staining in the immunoperoxidase method. J. Clin. Pathol., 30, 88–90PubMedCrossRefGoogle Scholar
  54. 54.
    Mepham, B. L., Frater, W. and Mitchell, B. S. (1979). The use of proteolytic enzymes to improve immunoglobulin staining by the PAP technique. Histochem. J. 11, 345–57PubMedCrossRefGoogle Scholar
  55. 55.
    Stein, H., Bonk, A., Tolksdorf, G., Lennert, K., Rodt, H. and Gerdes, J. (1980). Immunohistologic analysis of the organisation of normal lymphoid tissue and non-Hodgkin’s lymphomas. J. Histochem. Cytochem., 28, 746–60PubMedCrossRefGoogle Scholar
  56. 56.
    Warford, A. and Ketchin, G. S. (1986). The effect of fixation upon monoclonal cryostat immunohistochemistry. Med. Lab. Sci., 43, 128–34PubMedGoogle Scholar

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© Kluwer Academic Publishers 1990

Authors and Affiliations

  • B. L. Mepham
  • K. J. M. Britten

There are no affiliations available

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