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Antibody Elution Method for Multiple Immunohistochemistry on Primary Antibodies Raised in the Same Species and of the Same Subtype

Daniel Pirici, Laurentiu Mogoanta, Samir Kumar-Singh, Ionica Pirici, Claudiu Margaritescu, Cristina Simionescu and Radu Stanescu

Department of Histology (DP,LM) and Department of Pathology (CM,CS), University of Medicine and Pharmacy Craiova, Craiova, Romania; Neurodegenerative Brain Diseases Group, Vlaams Instituut voor Biotechnologie, Department of Molecular Genetics, Laboratory of Neurogenetics, Institute Born Bunge, University of Antwerp, Antwerp, Belgium (DP,SK-S); and Emergency County Hospital 1, Craiova, Romania (IP,RS)

Correspondence to: Daniel Pirici, MD, Department of Histology, University of Medicine and Pharmacy Craiova, Petru Rares Street 2, 200349 Craiova–Dolj, Romania. E-mail: daniel.pirici{at}ua.ac.be

Double or multiple antigen labeling in IHC classically relies on the existence of primary antibodies raised in different species or of different IgG isotypes to ensure the specific labeling with the secondary detection systems. However, suitable pairs of primary antibodies are not always available or the best choice (e.g., as diagnostic tools). During the last few years, several methods have been proposed to overcome this, but none of them offers the flexibility needed for reliable double or multiple enzymatic or fluorescent IHC. We present here a procedure that elutes the antibodies after a first round of immunolabeling, which, in combination with precipitation-based detection systems, allows multiple IHC rounds even for primary antibodies raised in the same species and IgG isotype. Compared with other proposed methods, this procedure ensures a reliable enzymatic or fluorescent staining without cross-reactivity and without loss of tissue antigenicity, thus offering a flexible tool for colocalization studies and pathological diagnosis. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 57:567–575, 2009)

Key Words: fluorescence microscopy • multiple enzymatic labeling • multiple immunofluorescent labeling • stripping buffer • precipitating substrates

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