The DAB Peroxidase Substrate Kit from Vector Laboratories is designed to simplify the process of staining through the use of premixed reagents. This kit includes four bottles, each containing approximately 15mL of each reagents - buffer, hydrogen peroxide, DAB, and a nickel solution. I have primarily used this kit for immunohistochemical staining using biotin-conjugated secondary antibodies. In this project, we are trying to identify puncta in the rat cochlear nucleus containing the neurotransmitter substance P. To do this, sections, in our case frozen 40-micron rat brain sections, are used. We use a standard immunocytochemistry protocol which includes using a biotin-conjugated secondary antibody followed by Vector Lab’s ABC (avidin-biotin complex) solution, which contains biotinylated peroxidase molecules. It is at this point in the procedure that the DAB kit is used.
I have been performing DAB immunocytochemistry on various tissues over a period of several years. Depending upon the particular DAB staining protocol used in the lab, after the incubation in the avidin-biotin solution, the sections to be stained are incubated in a solution of DAB (which I have used at a concentration of anywhere between 0.01% and 0.05%). Just prior to the DAB incubation it is necessary to add a small amount of hydrogen peroxide to the DAB solution so that the brown reaction product will develop. The final concentration of hydrogen peroxide in the DAB solutions I have used in the past vary between 0.01% and 0.75%, depending upon the exact concentration of the DAB solution and how quickly the researcher wishes the reaction to progress. With the Vectastain DAB kit there is no more inconsistency or guesswork in conducting the DAB reaction, as all solutions, when mixed as directed in terms of drops of stock solution per volume of working solution, will yield consistent and highly reliable concentrations of working solutions.
Clearly, the greatest advantage to using the Vectastain DAB kit is convenience: no pipetting of reagents, no dissolving of DAB tablets, which results in increased reliability and speed of preparation. Given that several dozen staining batches (using our protocol) can be performed with a single kit, and considering the time saved by not having to perform calculations, dilutions, or unnecessary handling of toxic/carcinogenic chemicals, the price of around $70.00 seems reasonable.
I can easily recommend this product, especially for an experimental procedure similar to that employed by our lab. Vector Labs also has substrate kits for alkaline phosphatase and glucose oxidase as well as an chemiluminescent substrates for peroxidase or alkaline phosphatase.
Ronald D. Griffith, Jr.
Research Associate I
University of Michigan