In Situ Localization of Nonenzymatic Peroxidase‐Like Activity of Tissue‐Bound Transition Metals

Lawrence M. Sayre1, Peggy L.R. Harris1, George Perry1, Mark A. Smith1

1 Case Western Reserve University, Cleveland, Ohio
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 17.4
DOI:  10.1002/0471140856.tx1704s20
Online Posting Date:  June, 2004
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Abnormalities in redox and/or transition metal homeostasis is characteristic of many pathological states. Here, we present protocols that can be used for the detection of both transition metals and redox state.

Keywords: iron; redox; DAB

PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Detection of Iron
  • Alternate Protocol 1: Detection of Redox‐Active Transition Metals
  • Support Protocol 1: Chelation of Metals for Controls
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1: Detection of Iron

  • Fresh tissue of interest
  • Fixative solution (see recipe)
  • 30%, 50%, 70%, 80%, 95%, and 100% (v/v) ethanol
  • Xylene
  • Paraffin
  • 50 mM Tris·Cl, pH 7.6 ( appendix 2A)
  • 7% (w/v) potassium ferrocyanide solution (see recipe), freshly prepared
  • DAB/H 2O 2 solution (for protocol 1; see recipe), freshly prepared
  • Permount mounting medium (e.g., Fisher)
  • Tissue‐embedding cassettes (Omnisette; Fisher)
  • Paraffin‐embedding system or machine (e.g., TBS ATPI Automated Tissue Processor; Fisher)
  • Microtome
  • Superfrost Plus microscope slides (Fisher)
  • Coplin jars
  • Humified chamber (storage container with sealable lid, lined with moistened paper towels and containing elevated surface on which to place slides) or container designed for immunohistochemical incubations
  • 3‐cc BD disposable syringes (Fisher)
  • 0.22‐µm Millex‐GS syringe filters (Millipore)
  • Coverslips

Alternate Protocol 1: Detection of Redox‐Active Transition Metals

  • Peroxide/methanol: add 4 ml of 30% hydrogen peroxide to 36 ml of 100% methanol; prepare fresh daily
  • DAB/H 2O 2 solution (for recipe; see recipe), freshly prepared
  • MBTH/o‐phenylenediamine/H 2O 2 solution (see recipe), freshly prepared

Support Protocol 1: Chelation of Metals for Controls

  • 0.1 M DEF stock: Weigh 65.68 mg deferoxamine (DEF; Sigma) and add 1 ml H 2O
  • 0.1 M DTPA stock: Weigh 39.33 mg diethylenetriaminepentaacetic acid (DTPA; Sigma) and add 1 ml H 2O
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Bancroft, J.D. and Stevens, A. (eds.) 1982. Theory and Practice of Histological Techniques, 2nd ed. Churchill Livingstone, New York.
   Brawer, J.R., Stein, R., Small, L., Cisse, S., and Schipper, H.M. 1994. Composition of Gomori‐positive inclusions in astrocytes of the hypothalamic arcuate nucleus. Anat. Record 240:407‐415.
   Connor, J.R., Menzies, S.M., St. Martin, S.M., and Mufson, E.J. 1990. Cellular distribution of transferrin, ferritin, and iron in normal and aged human brains. J. Neurosci. Res. 27:595‐611.
   Ngo, T.T. and Lenhoff, H.M. 1980. A sensitive and versatile chromogenic assay for peroxidase and peroxidase‐coupled reactions. Anal. Biochem. 105:389‐397.
   Nguyen‐Legros, J., Bizot, J., Bolesse, M., and Pulicani, J.‐P. 1980. Noir de diaminobenzidine: Une nouvelle methode histochimique de revelation du fer exogene. Histochem. 66:239‐244.
   Parmley, R.T., Spicer, S.S., and Alvarez, C.J. 1978. Ultrastructural localization of nonheme cellular iron with ferrocyanide. J. Histochem. Cytochem. 26:729‐741.
   Pearse, A.G.E. 1985. Histochemistry. Theoretical and Applied. Vol. 2. Analytical Technology, 4th ed., Churchill Livingstone, New York.
   Perls, M. 1867. Nachweis von Eisenoxyd in gewissen Pigmenten. Virchows Arch. Pathol. Anat. 39:42‐48.
   Sayre, L.M., Perry, G., Harris, P.L.R., Liu, Y., Schubert, K., and Smith, M.A. 2000. In situ oxidative catalysis by neurofibrillary tangles and plaques in Alzheimer's disease: A central role for bound transition metals. J. Neurochem. 74:270‐279.
   Schaermeyer, U. 1992. Localization of peroxidase activity in the retina and the retinal pigment epithelium of the Syrian golden hamster (Mesocricetus auratus). Comp. Biochem. Physiol. 103:139‐145.
   Schipper, H.M., Scarborough, D.E., Lechan, R.M., and Reichlin, S., 1990a. Gomori‐positive astrocytes in primary culture: Effects of in vitro age and cysteamine exposure. Dev. Brain Res. 54:71‐79.
   Schipper, H.M., Lechan, R.M., and Reichlin, S. 1990b. Glial peroxidase activity in the hypothalamic arcuate nucleus: Effects of estradiol valerate‐induced persistent estrus. Brain Res. 507:200‐207.
   Schipper, H.M., Vininsky, R., Brull, R., Small, L., and Brawer, J.R. 1998. Astrocyte mitochondria: A substrate for iron deposition in the aging rat substantia nigra. Exp. Neurol. 152:188‐196.
   Sternberger, L.A. 1986. Immunocytochemistry, 3rd ed. John Wiley & Sons, New York.
   Svensson, B.A., Rastad, J., and Westman, J. 1984. Endogenous peroxidase‐like activity in the feline dorsal column nuclei and spinal cord. Exp. Brain Res. 55:325‐332
   Thompson, S.W. 1966. Histochemical and Histopathological Methods, pp. 592‐594. Charles C. Thomas, Springfield, Ill.
   Trojanowski, J.Q., Obrocka, M.A., and 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. 31:1217‐1223.
PDF or HTML at Wiley Online Library