The animal research Kit (ARK) can be used in a multistep double staining method for human tissue specimens

The newly developed Animal Research Kit (ARK) offers a simple and economic way of biotinylating mouse primary antibodies for background-free immunosta ining of mouse and rat tissue specimens. Biotinylation involves the use of a biotinylated goat anti-mouse immunoglobulin Fab fragment mixed with a mou se primary antibody and subsequent blocking with normal mouse immunoglobuli n. Because a reliable immunoenzyme double staining procedure on human tissu e specimens with two unlabeled mouse primary antibodies of identical subcla ss is almost impossible, we have tested the performance of ARK biotinylatio n of one primary antibody in a multistep indirect/direct staining protocol. The multistep double staining procedure involved the subsequent applicatio n of an unlabeled mouse monoclonal antibody (MAb) 1 detected with an enzyme -labeled EnVision reagent, normal mouse serum for blocking, followed by a b iotinylated mouse MAb 2 and enzyme-labeled streptavidin. Alkaline phosphata se and peroxidase enzymatic activities were developed last. Double staining results obtained with an ARK biotinylated reagent were compared with a tru ly biotinylated reagent using N-hydroxy succinimide-biotin for conjugation. It appeared that both biotinylation procedures revealed identical double s taining results. Although a limited number of antibody combinations have be en tested, it is clear that this double staining procedure will be successf ul for many antibody pairs.