The VECTASTAIN ABC Kit is a very well-developed and versatile immunodetection system for frozen and embedded tissue sections from different animal species and blots (Western, Southern, Northern). The procedure for detection of tissue antigens using a biotinylated antibody and a pre-formed avidin:biotinylated enzyme complex (the “ABC” technique) was established by Su-Ming Hsu (J. Histochem. Cytochem. 29, 577, 1981) and further developed by Vector Laboratories. Many biotinylated enzyme molecules are crosslinked by avidin, forming three-dimensional complexes of heterogeneous size in the VECTASTAIN ABC Kit. In the 5-10 times more sensitive VECTASTAIN Elite ABC Kit, the complexes are smaller, very uniform and highly active. The kits offer low background and enhanced signal amplification as compared to conventional avidin/biotin conjugates.
The VECTASTAIN ABC Kits offer three different enzyme detection systems: VECTASTAIN ABC (peroxidase), VECTASTAIN ABC-AP (alkaline phosphatase), and VECTASTAIN ABC-GO (glucose oxidase). The standard peroxidase ABC kit contains 2 ml each of standard Reagent A (Avidin DH solution) and Reagent B (biotinylated enzyme). Other Vectastin peroxidase ABC kits (e.g. Vectastain ABC kit (mouseIgG), Vectastin ABC kit Human (IgG)) contain 3 ml of blocking serum (serum from the species in which the biotinylated secondary antibody was raised) and 1 ml of the biotinylated affinity-purified antibody. The solutions are provided in convenient dropper-bottles. Kits with stabilized, prediluted ‘ready-to-go’ solutions are also available. The VECTASTAIN ABC Kit contains enough reagents to stain 1000 to 2000 sections or develop about 50 blots (100 cm2), whereas the VECTASTAIN Elite ABC Kits contain sufficient reagents for 500 to 1000 slides or 25 100 cm2 blots. The kits are stable for several years when refrigerated.
For staining of mouse brain and spinal cord sections, I used the peroxidase detection systems. Since we used a variety of primary antibodies raised in different animal species (e.g. rabbit, sheep or goat), we ordered the specific biotinylated secondary antibodies and different animal sera, and used either the VECTASTAIN ABC or VECTASTAIN Elite ABC Standard Kit. Our antibodies raised in rabbits usually worked well with both kits. For mouse antibodies, which are generally more specific but less sensitive, the enhanced VECTASTAIN Elite ABC Kit worked better in most cases. Due to the more sensitive staining with the Elite kit, primary antibodies could be more dilute (2-3x), thereby reducing the antibody cost.
When I first tried a new primary antibody for staining, I used two different methods for detection in parallel in order to control for non-specific background staining, monitor the intensity of the antibody staining and assess the enhancement provided by the avidin/biotin system. After incubation with the primary antibody on 2 sections, I used one section for detection with a biotinylated secondary antibody and the avidin/biotinylated horseradish peroxidase (Vectastain ABC Kits) and applied only a horseradish peroxidase coupled secondary antibody (e.g. anti-rabbit IgG), without the avidin/biotin step to the other section. With some antibodies, I have found a slightly more brownish background with the avidin/biotin staining after DAB detection; this background was never strong enough to interfere with the specific staining of the antigen. We routinely use a slightly different protocol for immunostaining; our protocol calls for longer antibody incubations (overnight) and washes. Staining with the VECTASTAIN ABC Kits worked well with the lab protocol, and with the Vectastain protocol once the primary antibody dilution has been optimized. For mouse sections, we also successfully used the VECTASTAIN ABC Kit in combination with the M.O.M. Kit to reduce background staining with monoclonal mouse antibodies.
For me, it was important to make a note in the protocol to remember to prepare the working solution 30 min before applying it to the section. I liked that the stock solutions are provided in dropper-bottles; this not only makes it easy to prepare the working solution (to 5 ml (Elite) or 10 ml PBS, add 2 drops of solution A and 2 drops of solution B, mix), but also reduces the risk of contaminating solutions when opening bottles and pipetting the solutions.
For sensitive immunostaining using a peroxidase method I recommend the VECTASTAIN ABC Kits; they are easy to use, work well with high sensitivity and may reduce the cost for antibodies and also staining time. Vector Laboratories’ website contains not only the manuals for all of the kits, but also a troubleshooting table and another table listing different reagents useful for double staining.