The amplification of polymerized diaminobenzidine with physical developers: sensitizing effects of transition metal salts and sulphide

Abstract

SummaryAmplification of metal-complexed polymerized diaminobenzidine by two light-insensitive physical developers was systematically examined in a dot blot model system following either polymerizing diaminobenzidine in the presence of transition metal salts or applying the metal salts post-diaminobenzidine polymerization. The effect of sodium sulphide treatment on subsequent amplification was also investigated. Those metal-diaminobenzidine complexes that facilitated the most powerful amplification were subsequently tested in an immunohistochemical setting. The most dramatic amplification of polymerized diaminobenzidine was observed following its post-polymerization treatment with salts of platinum alone, or gold or vanadium with subsequent sulphide treatment, and allowed previously invisible quantities of polymerized diaminobenzidine to be clearly seen. Three other transition metal salts also improved the amplification of polymerized diaminobenzidine but to a lesser degree, namely nickel alone, and silver or rhodium with subsequent sulphide treatment. Sensitivity was comparable with the colloidal gold-silver amplification system.

In situ strategy for biomedical target localization via nanogold nucleation and secondary growth

“…4b ). The speculated reactivity between HAuCl 4 and DAB was verified by dot blotting on filter paper 20 (Fig. 4c ) and by dripping 0.05% HAuCl 4 into 0.02% DAB aqueous solution, which produced brown grains in a microtube.…”

“…The present in situ nanogold labeling was fortuitously developed on a laboratory bench, independent of previous studies. At the beginning of this study, we initially attempted to “enhance” the contrast of DAB deposition sites with 0.1% HAuCl 4 solution to convert the color signal into an electron-dense compound for electron microscopy 19 , 20 . It has been reported that exposure to gold chloride intensifies the electron density of DAB deposition sites 19 .…”

“…In immunohistochemical staining, DAB is known to be oxidized by hydrogen peroxide (H 2 O 2 ) in the presence of HRP that forms a brown deposition, representing the location of the HRP for light microscopy. Intensifications of DAB deposition sites have been reported with various heavy metallic ions as well as gold chloride 19 , 20 , 22 – 25 . The crucial binding ability between gold chloride and the immunochemical reaction product of DAB has been indicated by energy dispersive X (EDX)-ray analysis 25 .…”