, Volume 94, Issue 2, pp 217–223 | Cite as

Menadiol diphosphate, a new substrate for non-specific alkaline phosphatase in histochemistry and immunohistochemistry

  • A. Dikow
  • R. Gossrau
  • H. -G. Frank


Menadiol diphosphate was introduced as a new substrate for nonspecific alkaline phosphatase, following a search for new and less expensive substrates, which give a more sensitive response and are easily synthesized in the laboratory. Menadiol released by phosphatase action can be assayed by its reduction of tetrazolium salts, or it can be coupled with diazonium salts; alternatively, the phosphate can be trapped by metal ions. The synthesis and purification of menadiol diphosphate are described, and it was shown to be sufficiently stable for qualitative and semiquantitative histochemistry, as well as for the immunohistochemistry of enzymes and cytoskeletal proteins with nonspecific alkaline phosphatase as the enzyme label. For qualitative as well as semiquantitative histochemistry and immunohistochemistry, the best results were obtained by applying the method with nitro-blue tetrazolium (NBT) to acetone-chloroform pretreated cryostat sections. Tetranitro-blue tetrazolium (TNBT), benzothiazolylphthalhydrazidyl tetrazolium (BSPT) and various diazonium salts were less suitable. Fast Blue BB and VB produced satisfactory results. Ce3+ ions and the DAB−Ni−H2O2 procedure yielded better results than Ca2+ ions in the Co−(NH4)2S visualization method. The NBT method with menadiol diphosphate is superior to existing methods employing azo, azoindoxyl or tetrazolium salts and to metal precipitation methods. The Ce3+ technique and the NBT/menadiol diphosphate method give similar results, and appear to be of equal value. In qualitative histochemistry and immunohistochemistry the NBT/menadiol diphosphate method resulted in higher quantities of precisely localized stain. Semiquantitative histochemistry with minimal incubation revealed more favorable kinetics for the menadiol diphosphate method, especially when using NBT.


Tetrazolium Salt Diazonium Salt High Performance Thin Layer Chromatography Indoxyl Final Reaction Product 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Burstone MS (1962) Enzyme histochemistry and its application in the study of neoplasms. Academic Press, New York LondonGoogle Scholar
  2. Gossrau R (1983) Fluorescence histochemical detection of hydrolases in tissue sections and culture cells. Histochemistry 79:87–94PubMedCrossRefGoogle Scholar
  3. Gossrau R, Günther T, Merker H-J, Graf R (1988) Enhancement of maternal and fetal nephrotoxicity of salicylate by zinc deficiency. Morphological, enzyme histochemical and immunohistochemical studies. Histochemistry 89:81–90PubMedCrossRefGoogle Scholar
  4. Gutschmidt S, Lange U, Riecken EO (1980) Kinetic characterization of unspecific alkaline phosphatase at different sites of rat jejunum. A quantitative histochemical study. Histochemistry 69:189–202PubMedCrossRefGoogle Scholar
  5. Halbhuber K-J, Gossrau R, Möller U, Zimmermann N (1988) Light microscopic histochemistry of non-specific alkaline phosphatase using lanthanide-citrate complexes. Histochemistry 90:67–72PubMedCrossRefGoogle Scholar
  6. Hartel S, Gossrau R, Hanski C, Reutter W (1988) Dipeptidyl peptidase (DPP) IV in rat organs. Comparison of immunohistochemistry and activity histochemistry. Histochemistry 89:151–161PubMedCrossRefGoogle Scholar
  7. Lojda Z, Gossrau R, Schiebler TH (1979) Enzyme histochemistry. A laboratory manual. Springer, Berlin Heidelberg New YorkGoogle Scholar
  8. Luppa H, Ambrosius H, Storch W (1986) Immunreaktionen in der Histochemie. In: Graumann W, Neumann K (eds) Handbuch der Histochemie, Bd 4, Proteine 2. Teil. Fischer, Stuttgart New YorkGoogle Scholar
  9. Moss DW (1982) Isoenzymes. Chapman and Hall, London New YorkGoogle Scholar
  10. Raap AK (1986) Localization properties of fluorescence cytochemical enzyme procedures. Histochemistry 84:327–321CrossRefGoogle Scholar
  11. Ruhnke M (1990) Quantitativ-histochemische Untersuchungen der unspezifischen alkalischen Phosphatase und microsomalen Alanylaminopeptidase in der Decidua der Maus. Anat Anz [Suppl] (in press)Google Scholar
  12. Stahl E (1967) Dünnschicht-Chromatographie. Ein Laboratoriumshandbuch. Springer, Berlin Heidelberg New YorkGoogle Scholar
  13. Van Noorden CJF, Gossrau R (1990) Quantification in histochemistry and cytochemistry. In: Bach P, Baker JRJ (eds) Histochemistry and immunocytochemistry in toxicology. Chapman and Hall, London (in press)Google Scholar
  14. Van Noorden CJF, Jonges GN (1987) Quantification of the histochemical reaction for alkaline phosphatase using the indoxyltetranitro BT method. Histochem J 19:94–102PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 1990

Authors and Affiliations

  • A. Dikow
    • 1
  • R. Gossrau
    • 2
  • H. -G. Frank
    • 2
  1. 1.Bulgarian Academy of Sciences, Department of Cell Biology and MorphologyUlitza Akad. G. BontschevSofia
  2. 2.Department of AnatomyFree University of BerlinBerlin 33Federal Republic of Germany

Personalised recommendations