Skip to main content
SpringerLink
Log in

Sensitivity of various visualization methods for peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry

  • Papers
  • Published:
The Histochemical Journal Aims and scope Submit manuscript

Summary

Various chromogen protocols for visualizing peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry were compared with respect to their sensitivity. They were tested on tissue sections of human skeletal muscle and in an antigen spot test using antibodies against slow skeletal muscle myosin. The chromogens included 3-amino-9-ethylcarbazole (AEC), 3, 3′-diaminobenzidine (DAB),p-phenylenediamine-pyrocatechol (PPD-PC) and 4-chloro-1-naphthol (CN) in peroxidase histochemistry, and 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium salt (BCIP-NBT), BCIP-tetra nitro blue tetrazolium salt (TNBT) and various combinations of substituted naphthol phosphate-diazonium salt in alkaline phosphatase histochemistry. DAB, CN, and PPD-PC were also employed with imidazole and DAB in addition to Co2+ and Ni2+ ions. The results indicate that DAB-imidazole and DAB-Co2+ and Ni2+ ions are the most sensitive chromogen protocols for visualizing peroxidase activity. Although no large differences were found between the various chromogen protocols for visualizing alkaline phosphatase activity, the protocol BCIP-TNBT is especially recommended. Furthermore, the various chromogen protocols were evaluated as to stability of chromogen solutions and final precipitates, background staining, localization properties, and enhancement of enzyme activity.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Adams, J. C. (1981) Heavy metal intensification of DAB-based HRP reaction product.J. Histochem. Cytochem. 29, 775.

    Google Scholar 

  • Bondi, A., Chieregatti, G., Eusebi, V., Fulcheri, E. &Bussolati, G. (1982) The use of betagalactosidase as a tracer in immunocytochemistry.Histochemistry 76, 153–8.

    Google Scholar 

  • Bosman, F. T., Cramer-Knijnenburg, G. &Van Bergen Henegouw, J. (1983) Efficiency and sensitivity of indirect immunoperoxidase methods.Histochemistry 77, 185–94.

    Google Scholar 

  • Campbell, G. T. &Bhatnagar, A. S. (1976) Simultaneous visualization by light microscopy of two pituitary hormones in a single tissue section using a combination of indirect immunohistochemical methods.J. Histochem. Cytochem. 24, 448–52.

    Google Scholar 

  • De Jong, A. S. H., Van Vark, M., Albus-Lutter, Ch. E., Van Raamsdonk, W. &Voute, P. A. (1984) Myosin and myoglobin as tumor markers in the diagnosis of rhabdomyosarcoma. A comparative study.Am. J. Surg. Path. 8, 521–8.

    Google Scholar 

  • Graham, R. C. &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, 291–302.

    Google Scholar 

  • Graham, R. C., Ludholm, U. &Karnovsky, M. J. (1965) Cytochemical demonstration of peroxidase activity with 3-amino-9-ethyl-carbazole.J. Histochem. Cytochem. 13, 150–2.

    Google Scholar 

  • Hanker, J. S., Yates, P. E., Metz, C. B. &Rustioni, A. (1977) A new specific, sensitive and noncarcinogenic reagent for the demonstration of horseradish peroxidase.Histochem. J. 9, 789–92.

    Google Scholar 

  • Howard, A. N. &Wild, F. (1957) The reactions of diazonium compounds with amino acids and proteins.Biochem. J. 65, 651–9.

    Google Scholar 

  • Hsu, S. M. &Soban, E. (1982) Color modification of diaminobenzidine (DAB) precipitation by metallic ions and its application for double immunohistochemistry.J. Histochem. Cytochem. 30, 1079–82.

    Google Scholar 

  • Johansson, O. &Backman, J. (1983). Enhancement of immunoperoxidase staining using osmium tetroxide.J. Neurosci. Meth. 7, 185–93.

    Google Scholar 

  • Johnson, M. A., Polgar, J., Weightman, D. &Appleton, D. (1973). Data on the distribution of fibre types in thirty-six human muscles. An autopsy study.J. Neurol. Sci. 18, 111–29.

    Google Scholar 

  • Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of bacteriophage T4.Nature, Lond. 227, 680–5.

    Google Scholar 

  • Lowry, O. H., Rosebrough, N. J., Farr, A. L. &Randall, R. J. (1951) Protein measurement with the Folin phenol reagent.J. biol. Chem. 193, 265–75.

    Google Scholar 

  • Malik, N. J. &Daymon, M. E. (1982) Improved double immunoenzyme labelling using alkaline phosphatase and horseradish peroxidase.J. clin. Path. 35, 1092–4.

    Google Scholar 

  • Martin, T. L., Mufson, E. J. &Mesulam, M.-M. (1984) The light side of horseradish peroxidase histochemistry.J. Histochem. Cytochem. 32, 793.

    Google Scholar 

  • Mason, D. Y. &Sammons, R. (1978). Alkaline phosphatase and peroxidase for double immunoenzymatic labelling of cellular constituents.J. clin. Path. 31, 454–60.

    Google Scholar 

  • McGadey, J. (1970) A tetrazolium method for non-specific alkaline phosphatases.Histochemie 23, 180–4.

    Google Scholar 

  • Mesulam, M.-M. (1978) Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a noncarcinogenic blue reaction-product with superior sensitivity for visualizing neural afferents and efferents.J. Histochem. Cytochem. 26, 106–17.

    Google Scholar 

  • Morton, R. K. (1955) Some properties of alkaline phosphatase of cow's milk and calf intestinal mucosa.Biochem. J. 60, 573–82.

    Google Scholar 

  • Nakane, P. K. (1968) Simultaneous localization of multiple tissue antigens using the peroxidase-labeled antibody method: a study in pituitary glands of the rat.J. Histochem. Cytochem. 16, 557–60.

    Google Scholar 

  • Nakane, P. K. &Pierce, G. B. (1966). Enzyme-labeled antibodies: preparation and application for the localization of antigen.J. Histochem. Cytochem. 14, 929–31.

    Google Scholar 

  • Offer, G., Moos, C. &Starr, R. (1973) A new protein of the thick filaments of vertebrate skeletal myofibrils. Extraction, purification and characterization.J. molec. Biol. 74, 653–76.

    Google Scholar 

  • Raap, A. K. &Van Duijn, P. (1981) Enzyme-incorporated erythrocyte ghosts: a new model system for quantitative enzyme cytochemistry.J. Histochem. Cytochem. 29, 1418–24.

    Google Scholar 

  • Spreaficio, R., Cheema, S., Ellis, L. C. &Rustioni, A. (1982). On ‘comparison of horseradish peroxidase visualization methods’.J. Histochem. Cytochem. 30, 487–8.

    Google Scholar 

  • Straus, W. (1982). Imidazole increases the sensitivity of the cytochemical reaction for peroxidase with diaminobenzidine at a neutral pH.J. Histochem. Cytochem. 30, 491–3.

    Google Scholar 

  • Trojanovsky, J. Q., Obrocka, M. A. &Lee, V. M.-Y. (1983) A comparison of eight different chromogen protocols for the demonstration of immunoreactive neurofilaments or glial filaments in rat cerebellum using the peroxidase-antiperoxidase method and monoclonal antibodies.J. Histochem. Cytochem. 31, 1217–23.

    Google Scholar 

  • Van Duijn, P., Pascoe, E. &Van der Ploeg, M. (1967) Theoretical and experimental aspects of enzyme determination in a cytochemical model system of polyacrylamide films containing alkaline phosphatase.J. Histochem. Cytochem. 15, 631–45.

    Google Scholar 

  • Van Noorden, S. &Polak, J. M. (1983). Immunocytochemistry today. Techniques and practice. InImmunocytochemistry. Practical Applications in Pathology and Biology (edited byPolak, J. M. andVan Noorden, S.), pp. 11–42. Bristol, London and Boston: Wright PSG.

    Google Scholar 

  • Van Raamsdonk, W., Pool, C. W. &Heyting, C. (1977) Detection of antigens and antibodies by an immuno-peroxidase method applied on thin longitudinal sections of SDS-polyacrylamide gels.J. Immun. Meth. 17, 337–48.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pathology, Netherlands Cancer Institute (Antoni van Leeuwenhoekhuis), Amsterdam, The Netherlands

    A. S. H. De Jong & M. Van Kessel-Van Vark

  2. Department of Histochemistry and Cytochemistry, University of Leiden, Leiden, The Netherlands

    A. K. Raap

Authors
  1. A. S. H. De Jong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Van Kessel-Van Vark
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. K. Raap
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

De Jong, A.S.H., Van Kessel-Van Vark, M. & Raap, A.K. Sensitivity of various visualization methods for peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry. Histochem J 17, 1119–1130 (1985). https://doi.org/10.1007/BF01002537

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01002537

Keywords

Search

Navigation