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DOI: 10.1177/29.6.7252134 1981 29: 775J Histochem Cytochem

J C AdamsHeavy metal intensification of DAB-based HRP reaction product.

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- Jun 1, 1981Version of Record >>

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Received for publication January 16,1981; acceptedJanuary 23, 1981(LE 81-101

775

0022- 1554/8 l/08050 IS02.50The Journal of Histochemistry and CytochemistryCopyright 1981 by The Histochemical Society, Inc.

Vol. 29, No. 6, p. P5, 1981Printed in U.S.A.

Letters to the Editor

Heavy Metal Intensification of DAB-basedHRP Reaction Product

Despite the health hazard posed by diaminobenzidine (DAB), it re-mains the substrate ofchoice for several applications for demonstratingthe presence of horseradish peroxidase (HRP) in brain tissue. As asubstrate, tetramethylbenzidine (TMB) is more sensitive and it posesmuch less ofa health hazard (see, e.g., ref. 3), but the reaction productformed by TMB does not complex with osmium to form an electrondense substance. Moreover, the TMB-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 withHRP, such as when HRP has been iontophoresed into cells.

Several years ago it was noted that the DAB reaction productcould be made much darker than that of the Graham and Karnovski(2) procedure by performing the reaction in phosphate buffer in thepresence of cobalt chloride ( 1 ). Because cobalt precipitates readily inphosphate buffer, the tissue was first exposed to the cobalt in a Trisbuffer, excess cobalt rinsed from the tissue, and the reaction thencarried out in phosphate buffer. It has since been learned that thesensitivity of the reaction can further be enhanced by adding nickelsalts to the incubation medium. This communication describes thismodification and includes a much simpler and faster means of carryingout the reaction. The procedure is simplified by adding the metal saltsdirectly to the phosphate buffered DAB solution in concentrationsthat 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 (pH7.3) (DAB from some vendors does not readily dissolve at thispH. This problem can be remedied by dissolving the DAB ina lesser volume of H,O and then adding the appropriate buffer.)

2. While stirring the medium on a stir plate, slowly dropwise, add5.0 ml 1% cobalt chloride, then 4.0 ml 1% nickel ammoniumsulfate.

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. Mount 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 beingmore sensitive than the previous method ( 1 ), and has been used tolabel anterogradely transported HRP. It has the additional advantageof producing a black reaction product that should prove useful forimmunohistochemical applications. The reaction product is electrondense. 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. ADAMSDepartment of OtolaryngologyMedical University of South CarolinaCharleston, 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 MJ: The early stages of absorption ofinjected horseradish peroxidase in the proximal tubuler of mousekidney, ultrastructural cytochemistry by a new technique. J His-tochem Cytochem 14:291, 1966

3. Mesulam M, Rosene DL: Sensitivity in horseradish peroxidaseneurohistochemistry: a comparative and quantative study of ninemethods. J Histochem Cytochem 27:767, 1979

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