The VECTASTAIN Elite ABC system is the most sensitive avidin/biotin-based peroxidase system and is approximately 5 times more sensitive than the original VECTASTAIN ABC Kit. It is available without a biotinylated secondary antibody (Standard kit).
Advanced avidin/biotin technology: The Elite ABC complex is smaller, very uniform, and highly active, allowing more accessibility for binding to a biotinylated target
Cost effective: Higher sensitivity leads to lower cost per slide
Kit contains sufficient reagents to stain approximately 500-1000 sections
Immunohistochemistry / Immunocytochemistry, In situ hybridization, Blotting Applications, Elispot, ELISAs
I am performing IHC on tissue sections using a mouse IgG primary antibody. My detection system is a biotinylated anti-mouse IgG secondary antibody, a VECTASTAIN® Elite ABC peroxidase kit (PK-6100), in combination with a DAB substrate kit (SK-4100), and I do not see any staining. How do I know if the end detection reagents are working?
Make a fresh working solution of DAB substrate per instructions. Place a small volume (~1 mL) of this DAB substrate into a clean glass test tube. To this 1 ml aliquot, add one drop (~50 µL) of Reagent B only from the VECTASTAIN® Elite ABC kit. If the HRP enzyme is active and the DAB reacts with it, an immediate color change will be observed. This indicates the end detection reagents are working appropriately.
How do I choose which VECTASTAIN ABC Kit to use?
We offer a choice of either peroxidase or alkaline phosphatase based VECTASTAIN ABC kit detection systems. Selection of which enzyme system to use would be the first step. Most IHC applications involve detecting an unconjugated primary antibody. Consider the species in which the primary antibody is raised (e.g. rabbit), and then match to to corresponding species-specific VECTASTAIN ABC kit (e.g. VECTASTAIN ABC Rabbit IgG kit). If you already have a biotinylated target (i.e. biotinylated secondary antibody or primary antibody), then a standard VECTASTAIN ABC kit containing just the avidin/biotinylated enzyme reagents would be required. Note that only the VECTASTAIN Universal Elite PLUS ABC kit (PK-8200) contains a substrate.
Should the VECTASTAIN ABC kit only be stored at 4 degrees C? Can it be stored at -20 degrees C?
We do not recommend freezing the ABC kits. In particular, the ABC reagents exhibit a decrease in enzyme activity and lose some sensitivity when stored at - 20 °C for extended periods.
The VECTASTAIN ABC systems are extremely sensitive due to the form and number of active enzyme molecules associated with the preformed Avidin/Biotinylated enzyme Complex. This ABC complex takes advantage of two important properties of avidin: 1) an extraordinarily high affinity for biotin (over one million times higher than antibody for most antigens), and 2) four biotin-binding sites. These properties allow macromolecular complexes (ABCs) to be formed by mixing Avidin DH (Reagent A) with its paired biotinylated enzyme (Reagent B) prior to use. The ABC reagent, once formed, remains stable for many hours after formation and can be used for several days after preparation.
The VECTASTAIN ABC Reagent can be used to detect any molecule that is biotinylated. This property gives the avidin-biotin complex (ABC) method great versatility in the types of targets that can be detected as well as the types of applications in which it can be employed. Biotinylated primary antibodies, secondaries, lectins, neuronal tracers, nucleic acids, and ligands can be effectively visualized in applications such as:
Multiple labeling (Multiplex IHC)
Southern and northern blotting
In situ hybridization detection (ISH)
Enzyme immunoassays (ELISA)
All applications benefit from the high sensitivity, low background, reproducibility, and economy of the VECTASTAINABC system.
Choosing a VECTASTAIN ABC Kit
1) Choose an enzyme system:
The VECTASTAIN ABC Kits are available with a choice of two different enzyme systems
Peroxidase (ABC, Elite ABC, RTU or Kits)
Alkaline Phosphatase (ABC-AP)
2) Choose the biotinylated secondary antibody:
To detect a biotinylated target, you will need only the ABC Reagent contained in the Standard Kit, followed by an appropriate substrate.
To detect an unlabeled primary antibody, you will need a biotinylated secondary antibody that binds to the primary antibody species chosen, the ABC Reagent, and an appropriate substrate. For example, to detect a primary antibody made in rabbit, the appropriate choice is a VECTASTAIN ABC Kit designated “Rabbit IgG”.
For additional versatility and convenience, Vector Laboratories offers secondary antibody products that recognize more than one species of primary antibody. Our Universal Biotinylated Anti-Mouse/Rabbit IgG secondary antibody (Cat. No. BA-1400) is a cocktail of biotinylated anti-mouse IgG and anti-rabbit IgG, designed for use with both rabbit and mouse primary antibodies. This antibody should not be used to stain tissues from rodents or rabbits due to its reactivity with IgG present in the tissues of these species. The Universal Pan-Specific Anti- Mouse/Rabbit/Goat secondary antibody (Cat. No. BA-1300) (which is included in our VECTASTAIN Universal Kits) is a cocktail of biotinylated anti-mouse IgG, anti-rabbit IgG, and anti-goat IgG that recognizes mouse, rabbit, and goat primary antibodies. This secondary antibody should not be used to stain rat, mouse, or other rodent, rabbit, goat, bovine, or sheep tissue due to potential reactivity with endogenous IgG.
3) Choose a convenient format:
Finally, several kits are available in prediluted, stabilized formats for convenience and ease of use. See kits designated R.T.U. or Ready-To-Use.
Consider Species Cross-Reactivity
When choosing the optimal detection system for your application, it is important to consider not only the species of the primary antibody but also the species of the tissue under examination. If the species of the primary antibody and the species of the tissue are closely related (e.g. rat and mouse), the biotinylated secondary antibody may cross react with endogenous IgG in the tissue section leading to background staining. The following three options minimize background staining in these instances:
Biotinylate the primary antibody and detect with a Standard VECTASTAIN Elite ABC Kit, PK-6100 (only includes the avidin-biotin complex, or ABC reagent).
Use a biotinylated secondary antibody specifically adsorbed to remove cross-reacting antibodies of closely-related species (e.g. biotinylated anti-mouse IgG, rat adsorbed, Cat. No. BA-2001).
Use the M.O.M.® Immunodetection System for applications of mouse primary antibodies on mouse tissue.
After choosing the VECTASTAIN ABC Kit for your application, select a substrate that matches the enzyme system of the kit.