Stabilization of TMB reaction product for electron microscopic retrograde and anterograde fiber tracing

Brain Res Bull. 1985 Mar;14(3):277-81. doi: 10.1016/0361-9230(85)90093-0.


Use of the highly sensitive tetramethylbenzidine (TMB) method of horseradish peroxidase histochemistry for electron microscopy has been limited by the solubility of the reaction product in aqueous and alcoholic solutions. We have found that following the TMB reaction with a diaminobenzidine-cobalt (DAB-Co) step causes the TMB crystals to become coated with DAB-Co. The resultant reaction complex is insoluble, and easily localized using electron microscopy. By systematically varying the pH at which the TMB reaction is run, the size and shape of the reaction complex can be controlled. The pH 4.0 reaction complex was the most suitable for electron microscopic identification of labeled structures less than 1.0 micron in diameter (e.g., axon terminals).

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Benzidines*
  • Central Nervous System / anatomy & histology*
  • Cerebral Cortex / anatomy & histology
  • Chromogenic Compounds*
  • Gyrus Cinguli / anatomy & histology
  • Horseradish Peroxidase*
  • Male
  • Microscopy, Electron
  • Neural Pathways / anatomy & histology
  • Neuroanatomy / methods
  • Peroxidases*
  • Rats
  • Rats, Inbred Strains
  • Staining and Labeling / methods*


  • Benzidines
  • Chromogenic Compounds
  • 3,3',5,5'-tetramethylbenzidine
  • Horseradish Peroxidase
  • Peroxidases