TECHNICAL NOTE

The Animal Research Kit (ARK) Can Be Used in a Multistep Double Staining Method for Human Tissue Specimens

Correspondence to: Chris M. van der Loos, Academical Medical Centre, Dept. of Cardiovascular Pathology (H0-120), Meibergdreef 9, NL-1105 AZ Amsterdam, The Netherlands. E-mail: c.m.vanderloos@amc.uva.nl

The newly developed Animal Research Kit (ARK) offers a simple and economic way of biotinylating mouse primary antibodies for background-free immunostaining of mouse and rat tissue specimens. Biotinylation involves the use of a biotinylated goat anti-mouse immunoglobulin Fab fragment mixed with a mouse primary antibody and subsequent blocking with normal mouse immunoglobulin. Because a reliable immunoenzyme double staining procedure on human tissue specimens with two unlabeled mouse primary antibodies of identical subclass is almost impossible, we have tested the performance of ARK biotinylation of one primary antibody in a multistep indirect/direct staining protocol. The multistep double staining procedure involved the subsequent application of an unlabeled mouse monoclonal antibody (MAb) 1 detected with an enzyme-labeled EnVision reagent, normal mouse serum for blocking, followed by a biotinylated mouse MAb 2 and enzyme-labeled streptavidin. Alkaline phosphatase and peroxidase enzymatic activities were developed last. Double staining results obtained with an ARK biotinylated reagent were compared with a truly biotinylated reagent using N-hydroxy succinimide–biotin for conjugation. It appeared that both biotinylation procedures revealed identical double staining results. Although a limited number of antibody combinations have been tested, it is clear that this double staining procedure will be successful for many antibody pairs. (J Histochem Cytochem 48:1431–1437, 2000)

Key Words: human tissue, immunohistochemistry, double staining, EnVision, biotinylation, Fab fragment, Animal Research Kit, identical IgG subclass

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