The Vectastain Universal Elite ABC kit allows the visualization of antigens in tissue sections following labeling with a primary antibody. The visualization method is simple – once the primary antibody is bound to the antigen of interest, the biotinylated “universal” secondary antibody is added, forming a biotinylated antibody complex bound to the target antigen. By adding the Vectastain ABC reagent (Avidin and Biotinylated horseradish peroxidase [HRP] Complex), HRP is bound to the primary/secondary antibody complex. HRP enzyme activity can then be visualized by adding a chromogen substrate such as diaminobenzidine tetrachloride (DAB), which results in brown staining. The manufacturers also suggest the use of 3-amino-9-ethyl carbazole (AEC), which produces a red reaction product. We commonly use DAB and counterstain with Methyl Green, which produces a nice, light green contrast for negative cells. In addition to these commercially available substrates, Vector Laboratories also provides the option of purchasing unique peroxidase substrates – VIP (purple), SG (blue-gray) and NovaRED (red), enabling a variety of colorimetric reactions.
As with any immunohistochemical technique, sample preparation is paramount. We have used the Elite ABC kit on paraformaldehyde-fixed, paraffin embedded brain and colon sections and we have found little difference in the quality of signal achieved between tissues. The strength of this kit is that, given the strong affinity of avidin for biotin, the signal can be amplified many-fold over primary-secondary antibody interactions alone. I have tested our antibodies on tissues fixed with several different fixatives, including the above-mentioned paraformaldehyde, acetone, methanol, and Bouin’s fixative, and in each case color development using the ABC Elite kit was far superior to HRP-conjugated secondary antibodies alone. In at least one of the fixatives, no signal was visible unless the ABC kit was used.
The information which comes with the kit is quite detailed, and several protocols, considerations and references are provided, allowing optimization of the kit for individual experiments. One slight drawback is the need for the researcher to provide several chemicals, not included in the kit. However this is ameliorated by the fact that most of the reagents are readily available in the majority of labs, including hydrogen peroxide (to block endogenous peroxidase activity), PBS (washing), xylene and ethanol (if paraffin sections are used), a chromogen such as DAB, and naturally the primary antibody.
We have also incorporated an “antigen-retrieval” step (using either detergent, or boiling in a sodium citrate buffer), which allows better permeabilisation of tissues, and is an especially effective technique if the antigen of interest is intracellular. This antigen-retrieval step has no adverse effect on the ABC kit, and can also increase signal. We have found that background staining using the ABC kit can be problematic, although this can usually be minimized by decreasing primary antibody concentration, or increasing number/duration of washes.
In summary, the Vectastain Universal Elite ABC kit is a very powerful method for increasing signal intensity in immunohistochemistry. The range of troubleshooting advice and protocols available with the product information is, in my opinion, first-rate, and the kit is not overly expensive. However the secondary antibody provided in the kit, although called “universal”, is targeted against only mouse or rabbit IgG, although it may well work with antibodies raised in other species. Furthermore, DAB is a carcinogen, which must be handled carefully. Overall though, this is a kit I use often, and would have no hesitation in recommending to others.
Seb Dworkin
Graduate Student,
Peter MacCallum Cancer Centre