Antibody elution method for multiple immunohistochemistry on primary antibodies raised in the same species and of the same subtype

J Histochem Cytochem. 2009 Jun;57(6):567-75. doi: 10.1369/jhc.2009.953240. Epub 2009 Feb 16.


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 Please visit this article online to view these materials.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies*
  • Antigens, CD / immunology
  • Buffers
  • Collagen Type IV / immunology
  • Cross Reactions
  • Factor VIII / immunology
  • Fluorescent Dyes
  • Humans
  • Immunoglobulin G
  • Immunoglobulin Isotypes*
  • Immunohistochemistry / methods*
  • Ki-67 Antigen / immunology
  • Mice
  • Proliferating Cell Nuclear Antigen / immunology
  • Rabbits


  • Antibodies
  • Antigens, CD
  • Buffers
  • Collagen Type IV
  • Fluorescent Dyes
  • Immunoglobulin G
  • Immunoglobulin Isotypes
  • Ki-67 Antigen
  • Proliferating Cell Nuclear Antigen
  • Factor VIII