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

TLDR

Double or multiple antigen labeling in IHC traditionally requires primary antibodies from different species or IgG isotypes, but suitable pairs are often unavailable and existing methods lack the flexibility needed for reliable enzymatic or fluorescent IHC. The study introduces a method to elute antibodies after initial labeling to allow multiple IHC rounds with same‑species, same‑isotype primary antibodies. The procedure combines antibody elution with precipitation‑based detection systems to perform successive rounds of staining. Compared with other methods, this approach yields reliable enzymatic or fluorescent staining without cross‑reactivity or loss of antigenicity, providing a flexible tool for colocalization studies and pathological diagnosis. Online supplemental material is available at http://www.jhc.org.

Abstract

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.