Dear Nora, I have used several proprietary enhancements - which is a very easy, but somewhat expensive, way of enhancing DAB chromogen. It is especially easy if the enhancer is included in the staining kit. You may also use separately bought enhancer, as the one from DAKO - I have used it, and some others - it works fine.

Another way is, as Refik suggested, using metal salts (the same as in proprietary solutions). In early 1980s Hsu & colleagues pioneered DAB enhancement with several metal salts (he explored Ni, Co, Cu, Fe, Ag, Hg, Zn, Cr, Al, Pb, Pd and Ru). Only nickel chloride,(purplish-blue result), cobalt chloride (blue/blue-black result)  worked well without raising background, CuSO4 gave greyish-blue result but raised background. Remaining salts tested by Hsu either didn't enhance DAB. I have used both nickel chloride and cobalt chloride in the past on many occasions and found modified DAB stable, resisting organic solvents and contrasting the immunostaining very well against different nuclear counterstains. Hsu method called for adding 50mg DAB to 100 ml Tris buffered saline pH 7.6 and then 2 ml of metallic ion solution (1%). At the time of Hsu's experiments DAB was used in substantia (powder or tablets). For health & safety reasons powder is no longer used, but tablets are available. Easiest way is to use liquid DAB solution from the kit and enhance with a proportional solution of nickel chloride or cobalt chloride, depending on a volume of enhancer your are using).

I have put all this information about the early work on IHC enhancement, because you might find experimenting with DAB enhancement in the lab quite interesting. A word of caution - in consecutive experiments in your project you have to be very careful that you keep the same concentrations and timing. Good luck with your work.

Dear Nora,

I am not sure about this product from Abcam, but I would recommend you about my simple alternative (retro-style) Nickel intensified DAB method that definately increases your signal by many fold. It is simple, it is cheaper, and gives fantastic results everytime. For the recipe, details and references see my JCN paper (cited over 200 times) and PhD thesis (page 158).

best wishes, Refik

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Dear Nora, I have used several proprietary enhancements - which is a very easy, but somewhat expensive, way of enhancing DAB chromogen. It is especially easy if the enhancer is included in the staining kit. You may also use separately bought enhancer, as the one from DAKO - I have used it, and some others - it works fine.

Another way is, as Refik suggested, using metal salts (the same as in proprietary solutions). In early 1980s Hsu & colleagues pioneered DAB enhancement with several metal salts (he explored Ni, Co, Cu, Fe, Ag, Hg, Zn, Cr, Al, Pb, Pd and Ru). Only nickel chloride,(purplish-blue result), cobalt chloride (blue/blue-black result)  worked well without raising background, CuSO4 gave greyish-blue result but raised background. Remaining salts tested by Hsu either didn't enhance DAB. I have used both nickel chloride and cobalt chloride in the past on many occasions and found modified DAB stable, resisting organic solvents and contrasting the immunostaining very well against different nuclear counterstains. Hsu method called for adding 50mg DAB to 100 ml Tris buffered saline pH 7.6 and then 2 ml of metallic ion solution (1%). At the time of Hsu's experiments DAB was used in substantia (powder or tablets). For health & safety reasons powder is no longer used, but tablets are available. Easiest way is to use liquid DAB solution from the kit and enhance with a proportional solution of nickel chloride or cobalt chloride, depending on a volume of enhancer your are using).

I have put all this information about the early work on IHC enhancement, because you might find experimenting with DAB enhancement in the lab quite interesting. A word of caution - in consecutive experiments in your project you have to be very careful that you keep the same concentrations and timing. Good luck with your work.

Dear Refik,

Thank you for your quick reply and the attached sources. "Retro-style" is sometimes the most reliable way to go, will give it a go. 

Dear Maria, 

Thank you for the thorough answer, very intersting information about these pioneer experiments. I am surprised again and again by just how many things could one optimize while setting up an IHC staining. :) The timing of DAB incubation is defnintely a critical factor, thanks for the reminder. I think in the first round I will give the commercial enhancer a go, since it is available in our lab, but if the results are not that much better then nickel will be my next choice, just like in Refik's protocol. 

Thank you both for contributing!

Thanks all of you for the question (Nora) and for a very generous comments (Refik) and informative answer (Maria). This is what i was exactly in need.