Simultaneous visualization of multiple antigens with tyramide signal amplification using antibodies from the same species

J Histochem Cytochem. 2007 Jun;55(6):545-54. doi: 10.1369/jhc.6A7134.2007. Epub 2007 Jan 22.


After immunohistochemistry (IHC) began to be used routinely, a number of investigators worked on methods for staining multiple molecules in the same tissue sections or cells. Achieving this goal was not easy, however. One reason for this is that the majority of primary antibodies used in IHC reactions are raised in rabbits, and recognizing signals from two different rabbit antibodies is not trivial. Thus, all of the protocols described to date have serious limitations. Here we report a simple, quick, and inexpensive solution to the problem. It has two major advantages over existing methods. First, by using antibodies from the same host, two or more antigens can be visualized in the same section with commercially available fluorescent dyes. Second, because the technique relies on signal amplification, both rare and abundant antigens can be detected.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies / chemistry
  • Antibodies / immunology*
  • Antibody Specificity / immunology
  • Antigens / analysis
  • Antigens / immunology*
  • Cross Reactions / immunology
  • Fluorescein-5-isothiocyanate / chemistry
  • Fluorescent Dyes / chemistry
  • Glial Fibrillary Acidic Protein / analysis
  • Glial Fibrillary Acidic Protein / immunology
  • Goats
  • Immunoglobulin G / immunology
  • Immunohistochemistry / methods*
  • Male
  • Mice
  • Microwaves
  • Peroxidase / chemistry
  • Rabbits
  • Rats
  • Reproducibility of Results
  • Species Specificity
  • Tyramine / chemistry*
  • Tyrosine 3-Monooxygenase / analysis
  • Tyrosine 3-Monooxygenase / immunology
  • Vasopressins / analysis
  • Vasopressins / immunology


  • Antibodies
  • Antigens
  • Fluorescent Dyes
  • Glial Fibrillary Acidic Protein
  • Immunoglobulin G
  • Vasopressins
  • Peroxidase
  • Tyrosine 3-Monooxygenase
  • Fluorescein-5-isothiocyanate
  • Tyramine