Immunohistochemistry is often associated with excessive troubleshooting due to the extensive nature of the protocols involved. In addition, the antibodies needed for many protocols are often available only in limited quantities and/or have high prices. Resources are often wasted on preliminary experiments before reproducible results are attained. As a result, researchers are constantly striving to find reagents that allow conservation of expensive reagents, such as primary and secondary antibodies, both during and after experimental optimization. I have tested several immunohistochemical kits and have found the Vectastain ABC-Alkaline Phosphatase Kit from Vector Labs to be the most cost effective and easy to use.
When I initially tried this kit, I used dilutions recommended for individual antibodies, other protocols and other kits. However, I quickly found that due to superior signal amplification using the Vectastain kit, I was able to detect lower amounts of antigen in my samples, while cutting primary antibody dilutions down drastically. The Vectastain ABC-AP Kit allowed me to use as little as 0.1 ug/ml of primary antibody compared to other alternatives that had routinely required up to 10 ug/ml (the appropriate dilutions must be determined for each primary antibody). Experience has enabled me to use a primary antibody starting concentration of 0.2 ug/ml on paraffin imbedded samples, employing this kit in conjunction with Vector Blue Alkaline Phosphatase Substrate (also available from Vector Labs). Obviously, decreasing my starting dilution to such a low level has allowed me to conserve large amounts of valuable primary antibody without compromising my results.
Incubation periods necessary for reproducible results with the Vectastain kit are flexible and can be performed at room temperature. Conveniently, I have found that the primary antibody incubation can be as little as one hour if I am interested in doing the entire staining procedure in one day. If more time is needed, the primary antibody can also incubate for a few hours to overnight without an increase in background signal. If the overnight incubation period is used, which I now routinely perform, I extend the wash time following removal of the primary antibody to keep the background as low as possible. Like the primary antibody incubation, the secondary antibody incubation is also flexible and can range from 30 minutes to 1 hour.
The Vectastain ABC-AP Kit includes blocking serum, biotinylated antibody, Vectastain ABC-AP reagent and convenient mixing bottles. The provided reagents can easily be made into their working concentrations by adding the indicated number of drops to a basic 10 mM sodium phosphate solution (pH 7.5) 0.9% saline (PBS). I have found that mixing the reagents in another tube enables me to maximize the provided solutions because I am able to add exactly 150 ul of the working solutions to each sample. The bottles provided in the kit also work well because they dispense approximately 50 ul per drop.
My experience with this kit has demonstrated that it works with several different fixation protocols and incubation periods. Furthermore, the substrates offered by Vector Labs give the researcher a range of color choices for staining, thus different tissues can be stained to optimize visualization. So far, I have found the Vector Blue substrate to work best, due to the clear color development and low background. Together, the ease of use of the Vectastain ABC-AP Kit and the minimal amount of primary antibody needed for clearly reproducible results make it a good choice for any laboratory interested in performing immunohistochemistry.
Amy Bellmeyer, M.S.
Research Technician
Northwestern University
Division of Pulmonary and Critical Care Medicine