Summary
Quantitative techniques in immunohistochemistry are needed, but they are rarely applied because of doubtful reproducibility. We have developed a method for the detection of collagen types I and III in situ. The method applied was a two-step immuno-alkaline phosphatase technique with visualization of the end-product with Fast Red. The staining intensity was measured with a microdensitometer and the results expressed as ratios. The method yielded results that were unaffected by variations in tissue section thickness but which were proportionally related to time and antigen concentrations. Leiomyoma tissue, with a ratio of collagen types I and III of approximately 1.0, was used to establish the appropriate dilutions of the antibodies, thus assuring identical optical densities. By having the leiomyoma tissue sections incubated together with the heart tissue specimens, leiomyoma tissue was also helpful in correcting deviations from the 1.0 ratio. Accurate measurements of collagen type I/III ratios in normal human heart specimens were obtained with the present quantitative immunohistochemical technique.
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References
Borgers, M. (1973) The cytochemical application of new potent inhibitors of alkaline phosphatase. J. Histochem. Cytochem. 21, 812–24.
Brandtzaeg, P. (1982) Tissue preparation methods for immunohistochemistry. In Techniques in Immunohistochemistry (edited by Bullock, G. R. & Petrusz, P.) Vol. 1. p. 1–75. London: Academic Press.
Burnstone, M. S. (1962) Enzyme Histochemistry, p. 267. London: Academic Press.
Butcher, R. G. (1984) Reaction rate studies of glucose-6-phosphate dehydrogenase in rat tracheal epithelium: effect of section thickness. Histochemistry 81, 567–72.
Champelovier, P., Seigneurin, D., Christophe, P. & Kolodie, L. (1991) Quantification by image analysis of immunocyto-chemical reactions: application for determination of lysozyme content in individual smeared cells. J. Histochem. Cytochem. 39, 31–6.
Fritz, P., Mulhaupt, H., Hoenes, J., Lutz, D., Doerrer, R., Schwarzmann, P. & Tczek, H. V. (1992) Quantitative immunohistochemistry. Theoretical background and its application in biology and surgical pathway. Prog. Histochem. Cytochem. 24, No. 3, Stuttgart, Germany: Gustav Fisher Verlag.
James, J., Bosch, K. S., Aronson, D. C. & Houtkooper, J. M. (1990) Sirus Red histophotometry and spectrophotometry of sections in the assessment of the collagen content of liver tissue and its application in growing rat liver. Liver 10, 1–5.
Junqueira, L. C. U., Montes, G. S. & Sanches, E. M. (1982) The influence of tissue section thickness on the study of collagen by the picosirius-polarization method. Histochemistry 74, 153–6.
Laurent, G. L., Cockerill, P., Mcanulty, R. J. & Hasterings, J. R. B. (1981) A simplified method for quantification of the relative amounts of type I and III collagen in small tissue samples. Anal. Biochem. 113, 301–12.
Leunissen, J. L. M. & Van DePlas, P. (1993) Ultrasmall gold probes and cryo-ultramicrotomy. In Immuno-gold Electron Microscopy in Virus Diagnosis and Research (edited by Hyatt, A. D. & Eaton, B. T.). p. 327–48. Boca Raton, Florida: CRC Press.
Millar, D. A. & Williams, E. D. (1982) A step-wedge standard for the quantification of immunoperoxidase techniques. Histochem. J. 14, 609–20.
Nibbering, P. H. & VanFurth, R. (1987) Microphotometric quantitation of the reaction product of several indirect immunoperoxidase methods demonstrating monoclonal antibody binding to antigens immunobilized on nitrocellulose. J. Histochem. Cytochem. 35, 1425–31.
Nibbering, P. H., Marijnen, J. G. J., Raap, A. K., Leijh, P. C. J. & VanFurth, R. (1986) Quantitative study of enzyme immunocytochemical reactions performed with enzyme conjugates immunobilized on nitrocellulose. Histochemistry 84, 538–43.
Stierhof, Y-D., Schwarz, H. & Hermann, F. (1986) Transverse sectioning of plastic embedded immuno-labeled cryosections: morphology and permeability to protein A-gold complexes. J. Ultrastruc. Mol. Struc. Res. 97, 187–96.
Trelstad, R. L., Catanese, V. M. & Rubin, D. F. (1976) Collagen fractionation: separation of native types I, II and III by differential precipitation. Anal. Biochem. 71, 114–8.
VanNoorden, C. J. F. (1989) Principles of cytophotometry in enzyme histochemistry and validity of the reactions. Acta Histochemica Suppl. 37, 21–35.
VanNoorden, C. J. F. & Butcher, R. G. (1991) Quantitative enzyme histochemistry. In Histochemistry Theoretical and Applied. 4th edn (edited by Stoward, P. J. & Pearse, A. G. E.) p. 355–432. Vol. 3. Edinburgh: Churchill Livingstone.
VanNoorden, C. J. F. & Jonges, G. N. (1987) Quantification of the histochemical reaction for alkaline phosphatase activity using the indoxyl-tetranitro BT method. Histochem. J. 19, 94–102.
VanNoorden, C. J. F. & Vogels, I. M. C. (1989) Polyvinylalcohol and other tissue protectants in enzyme histochemistry: a consumer's guide. Histochem. J. 21, 373–9.
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Van Der Loos, C.M., Marijianowski, M.M.H. & Becker, A.E. Quantification in immunohistochemistry: the measurement of the ratios of collagen types I and III. Histochem J 26, 347–354 (1994). https://doi.org/10.1007/BF00157768
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DOI: https://doi.org/10.1007/BF00157768