Vectastain Universal Elite ABC Kit From Vector Laboratories

Investigation of cancer-specific molecules is a key area of research in modern biology. For cancer related studies, analysis of human tumor tissue for protein expression pattern is indispensable. The amount of tissue available is always a limitation considering the number of biochemical analyses to be carried out. Immunohistochemistry is a powerful technique for identification of specific proteins and their cellular localization within a cell. Immunohistochemistry also requires less tissue for analysis as compared to other protein techniques, like Western blotting. Of a number of kits available in the research market, we found the Vectastain Universal Elite ABC Kit to be suitable for all our immunohistochemical studies; it is sensitive, economical and reliable.

The Vectastain Elite ABC Kit has evolved from the ABC system and is more sensitive than the Vectastain ABC peroxidase system, but without an increase in background. One of the biggest advantages of immunohistochemistry is that studies can be performed on archival samples. The kit can also be used for Western blotting; however, we have other well-standardized techniques for Western blotting and do not use this system for Westerns as it does not prove to be cost effective.

The kit consists of (1) Blocking serum (Horse serum), (2) Biotinylated universal antibody and (3) Vectastain elite ABC reagent A/B.

When using paraffin embedded sections, we perform the antigen retrieval step followed by inactivation of endogenous peroxidase by incubating in 0.3% H₂O₂. In the case of frozen sections, the antigen retrieval step is omitted. In order to save on costly antibodies and reagents, we encircle the tissue section by a hydrophobic barrier pen known as ImmEdge pen from Vector Labs; this helps to minimize the usage of all the reagents and prevents mixing of two solutions on a slide.

50 ul of blocking serum is added to 5 ml of buffer and used for blocking the tissue section. Blocking of the section is performed for 1 hour at room temperature. Primary antibody of different dilutions is applied on the tissue sections for 1 hour followed by washing with buffer for 5 minutes X 3. 100 ul of biotinylated secondary antibody is added in 5 ml of buffer and is applied on tissue sections and incubated for an hour. This is a good reagent which binds antibodies of both rabbit and mouse origin and hence, is called the “universal” biotinylated secondary antibody. The best part of this kit is that we do not need to optimize the dilution of either the secondary or the tertiary antibody. Tissue sections are washed for 5 minutes X 3 and then are incubated with tertiary reagent which consists of preformed avidin and a biotinylated horseradish peroxidase macromolecular complex (prepared by addition of 100 ul of reagent A and 100 ul of reagent B in 5 ml of buffer). This complex is especially optimized to form ideal complexes for immunoperoxidase staining. After washing, development is carried out by diaminobenxidine tetrahydrochloride (DAB) and H₂O₂ which gives an insoluble reddish brown precipitate on the sections. The results with this kit so far have been superior and incomparable to the conventional methods of immunohistochemistry (i.e. using different secondary antibodies of different companies).

We perform immunohistochemistry studies for identification of intermediate filament and associated proteins in normal, pre-malignant and malignant tissues in order to establish them as markers for cancer. So far, our lab has published many papers based on this kit from Vector Laboratories. We have found the Vectastain Elite ABC Kit to be one of the best kits for specific detection of molecules in cancer tissues. It will definitely aid in establishing certain proteins as markers for early detection and prognosis of cancer. I will recommend it to any researcher using immunohistochemistry as a tool for cancer biology or biology in general.