PMID: 6159394Nov 1, 1980Paper

Additional factors influencing sensitivity in the tetramethyl benzidine method for horseradish peroxidase neurohistochemistry

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society
M M MesulamE J Mufson

Abstract

In experiments that use horseradish peroxidase (HRP) and tetramethyl benzidine (TMB) for tracing neural connections, the activity of tissue-bound enzyme as well as the stability of the resultant reaction product are influenced by the duration of storage, the composition of the storage medium, the type of counterstaining and even the details of histological dehydration. Furthermore, the conditions for preserving HRP activity are very different from those necessary for preserving the stability of the tetramethyl benzidine (TMB) reaction product. Thus, tissue-bound HRP activity is stable at a neutral pH, while a much lower pH, around 3.3, is required for preserving the stability of the TMB reaction product. Recent evidence indicates that the stabilization bath in sodium nitroferricyanide that was previously recommended is not necessary. However, gradual dehydration of mounted sections is essential for long-term stability. Excessive counterstaining and excessive dehydration interfere with the detection of reaction product. These considerations are pertinent to experiments using free HRP as well as to those where the enzyme has been conjugated to wheat germ agglutinin.

Citations

Dec 1, 1982·Anatomy and Embryology·P G Clarke
Mar 15, 1982·Experientia·C F Hinrichsen, A Ryan
Oct 28, 1982·Brain Research·D T Hess
Mar 4, 1985·Brain Research·J Ullán
Oct 2, 1989·Brain Research·R M Burde, F Williams
Sep 1, 1987·Journal of the Neurological Sciences·H AldskogiusL Thomander
Jan 1, 1982·Vision Research·T FitzGibbon
Jan 1, 1988·Vision Research·M L ManningB Fenelon
Sep 1, 1995·Journal of Neuroscience Methods·N Todorova, G S Rodziewicz
Jul 1, 1983·Journal of the Autonomic Nervous System·J E Norvell, J M Anderson
Dec 1, 1988·Brain Research. Developmental Brain Research·A IrikiY Nakamura
Feb 1, 1993·Neuroscience Research·N KudoN Okado

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