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Journal of Histochemistry & Cytochemistry
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DOI: 10.1177/29.6.7252134
1981 29: 775 J Histochem Cytochem
J C Adams
Heavy metal intensification of DAB-based HRP reaction product.

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Received for publication January 16,
1981; acceptedJanuary 23, 1981
(LE 81-101
775
0022- 1 554/8 l/080 50 IS02.50
The Journal of Histochemistry and Cytochemistry
Copyright 1981 by The Histochemical Society, Inc.
Vol. 29, No. 6, p. P5, 1981
Printed in U.S.A.
Letters to the Editor
Heavy M etal Intensification of DAB-based
HRP Reaction Product
Despite the health hazard posed by diaminobenzidine (DAB), it re-
mains the substrate ofchoice for several applications for demonstrating
the presence of horseradish peroxidase (HRP) in brain tissue. As a
substrate, tetramethylbenzidine (TM B) is more sensitive and it poses
much less ofa health hazard (see, e.g., ref. 3), but the reaction product
formed by TM B does not complex with osmium to form an electron
dense substance. M oreover, the TM B-based reaction product is de-
cidedly inferior to DAB-based reaction product for immunohisto-
chemistry, and for delineating cell process that contains a lot of HRP.
Consequently DAB is still used for applications where electron mi-
croscopy is to be done and where cells are extremely well filled with
HRP, such as when HRP has been iontophoresed into cells.
Several years ago it was noted that the DAB reaction product
could be made much darker than that of the Graham and Karnovski
(2) procedure by performing the reaction in phosphate buffer in the
presence of cobalt chloride ( 1 ). Because cobalt precipitates readily in
phosphate buffer, the tissue was first exposed to the cobalt in a Tris
buffer, excess cobalt rinsed from the tissue, and the reaction then
carried out in phosphate buffer. It has since been learned that the
sensitivity of the reaction can further be enhanced by adding nickel
salts to the incubation medium. This communication describes this
modification and includes a much simpler and faster means of carrying
out the reaction. The procedure is simplified by adding the metal salts
directly to the phosphate buffered DAB solution in concentrations
that barely saturate the medium with the salts. It is done as follows:
1 . Dissolve 1 00 mg DAB in 200 ml 0. 1 M phosphate buffer (pH
7.3) (DAB from some vendors does not readily dissolve at this
pH. This problem can be remedied by dissolving the DAB in
a lesser volume of H,O and then adding the appropriate buffer.)
2. W hile stirring the medium on a stir plate, slowly dropwise, add
5.0 ml 1% cobalt chloride, then 4.0 ml 1% nickel ammonium
sulfate.
3. Incubate tissue in this mixture for 15-20 mm.
4. Add 0.66 ml 3% H,O2.
5. Incubate for an additional 10-15 mm.
6. Rinse sections in 0. 1 M phosphate buffer.
7. M ount sections and proceed as usual.
This procedure eliminates the presoak in Tris buffered cobalt chlo-
ride and the subsequent rinses. It has the added advantage of being
more sensitive than the previous method ( 1 ), and has been used to
label anterogradely transported HRP. It has the additional advantage
of producing a black reaction product that should prove useful for
immunohistochemical applications. The reaction product is electron
dense. Consequently, despite the use of DAB and its inherent risks,
the method should prove useful for a variety of applications.
Acknowledgment
This technique was developed uhile the author uas a StaffFellou at LNO,
NINCDS. NIH.
Literature Cited
JOE C. ADAM S
Department of Otolaryngology
M edical University of South Carolina
Charleston, South Carolina 29403
I . Adams JC: Technical consideration on the use of horseradish per-
oxidase as a neuronal marker. Neuroscience 2:141, 1977
2. Graham RC Jr, Karnovsky M J: The early stages of absorption of
injected horseradish peroxidase in the proximal tubuler of mouse
kidney, ultrastructural cytochemistry by a new technique. J His-
tochem Cytochem 14:291, 1966
3. M esulam M , Rosene DL: Sensitivity in horseradish peroxidase
neurohistochemistry: a comparative and quantative study of nine
methods. J Histochem Cytochem 27:767, 1979
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