Summary
Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneusly demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3–7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol. These results indicate that bone AlP and AcP activities can be demonstrated simultaneously in the same section using a simple tissue preparation technique and that the activities are retained in tissues fixed and/or stored in acetone, 70% ethanol or GMA, but are differentially inactivated by other fixatives studied, and by EDTA, formic acid-citrate, and MMA embedding.
Similar content being viewed by others
Abbreviations
- AcP:
-
acid phosphatase
- AlP:
-
alkaline phosphatase
- GMA:
-
glycol methacrylate
- MMA:
-
methyl methacrylate
- EDTA:
-
ethylenediaminetetraacetic acid
References
Baneroft JD (1975) Histochemical techniques, 2nd edn. Butter-worths, London, pp 223–229
Barka T, Anderson PJ (1962) Histochemical methods for acid phosphatase using hexazonium parasosanilin as coupler. J Histochem Cytochem 10:741–753
Borgers M (1973) The cytochemical application of new potent inhibitors of alkaline phosphatases. J Histochem Cytochem 21:812–824
Burstone MS (1959) Histochemical demonstration of acid phosphatase activity in osteoclasts. J Histochem Cytochem 7:39–41
Burstone MS (1960a) Hydrolytic enzymes in dentinogenesis and osteogenesis. In: Sognnaes RF (ed) Calcification in biological systems, American Association for the Advancement of Science, Washington, DC, pp 217–243
Burstone MS (1960b) Histochemical observations on enzymatic processes in bones and teeth. Ann NY Acad Sci 85:431–444
Burstone MS (1962) Enzyme histochemistry and its application in the study of neoplasms. Academic Press, New York, pp 160–292
Changus GW (1957) Convenient histochemical phosphatase technique. Cancer 10:560–562
Davis BJ, Ornstein L (1959) High resolution enzyme localization with a new diazo reagent, “hexazonium pararosaniline.” J Histochem Cytochem 7:297–298
Doyle WL (1948) Effects of dehydrating agents on phosphatases in the lymphatic nodules of the rabbit appendix. Proc Soc Exp Biol Med 69:43–44
Fallon MD, Teitelbaum SL (1981) A simple procedure for the rapid histologic diagnosis of metabolic bone disease. Calcif Tissue Int 33:281–283
Farley JR, Ivey JL, Baylink DJ (1980) Human skeletal alkaline phosphatase — kinetic studies including pH dependence and inhibition by theophylline. J Biol Chem 255:4680–4686
Farley JR, Chesnut CH III, Baylink DJ (1981) Improved method for quantitative determination in seum of alkaline phosphatase of skeletal origin. Clin Chem 27:2002–2007
Farley JR, Puzas JE, Baylink DJ (1982) Effect of skeletal alkaline phosphatase inhibitors on bone cell proliferation in vitro. Min Electrolyle Metab 7:316–323
Gomori G (1952) Microscopic histochemistry: Principles and practice. University of Chicago Press, Chicago, Illinois
Greep RO, Fischer CJ, Morse A (1947) Histochemical demonstration of alkaline phosphatase in decalcified dental and osseous tissues. Science 105:666
Hammarström LE, Hanker JS, Toverud SU (1971) Cellular differences in acid phosphatase isoenzymes in bone and teeth. Clin Orthop Rel Res 78:151–167
Horobin RW (1982) Histochemistry: An explanatory outline of histochemistry and biophysical staining. Gustav Fischer, New York Stuttgart pp 187–206
Humason GL (1967) Animal tissue techniques, 2nd edn. Freeman & Co, San Francisco, pp 3–32
Kiernan JA (1981) Histological and histochemical methods. Theory and practice. Pergamon Press, New York, pp 8–24
Liu CC, Rader JI, Gruber H, Baylink DJ (1982) Acute reduction in osteoclast number during bone repletion. Metab Bone Dis Relat Res 4:201–209
Liu CC, Baylink DJ (1984) Differential response in alveolar bone osteoclasts residing at two different bone sites. Calfic Tissue Int 36:182–188
Meunier PJ (1983) Histomorphometry of the skeleton. In: Peck WA (ed) Bone and mineral research, Annual 1. Excerpta Medica, Amsterdam, pp 191–222
Mowry RW (1949) A comparison of technical procedures for the histochemical preservation of alkaline phosphatase. Bull Int Assoc Med Mus 30:95–98
Pearse AGE (1968) Histochemistry: Theoretical and applied, vol 1, 3rd edn. Little, Brown & Co, Boston
Robison R (1923) The possible significance of hexosephosphoric esters in ossification. Biochem J 17:286–293
Schajowicz F, Cabrini RL (1959) Histochemical demonstration of acid phosphatase in hard tissues. Stain Technol 34:59–64
Seligman AM, Chauncey HH, Nachlas MM (1951) Effect of formalin fixation on the activity of five enzymes of rat liver. Stain Technol 26:19–23
Stafford RO, Atkinson WB (1948) Effect of acetone and alcohol fixation and paraffin embedding on activity of acid and alkaline phosphatases in rat tissues. Science 107:279–281
Te Velde J, Burkhardt R, Kleiverda K, Leenheers-Binnendijk L, Sommerfeld W (1977) Methyl methacrylate as an embedding medium in histopathology. Histopathology 1:319–330
Thompson ER, Baylink DJ, Wergedal JE (1975) Increases in number and size of osteoclasts in response to calcium or phosphorus deficiency in the rat. Endocrinology 97:283–289
Troyer H, Nusbickel FR (1975) Enzyme histochemistry of undecalcified bone and cartilage embedded in glycol methacrylate. Acta Histochem 53 (Suppl):198–202
Weber R, Weber J, Niehus B (1961) Activation of acid phosphatase by versene. Helv Physiol Pharmacol Acta 19:97–102
Wergedal JE, Baylink DJ (1969) Distribution of acid and alkaline phosphatase activity in undemineralized sections of the rat tibial diaphysis. J Histochem Cytochem 17:799–806
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Liu, C., Sanghvi, R., Burnell, J.M. et al. Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities. Histochemistry 86, 559–565 (1987). https://doi.org/10.1007/BF00489547
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00489547