UNIT 2.12 The Importance of Titrating Antibodies for Immunocytochemical Methods

  1. Gloria E. Hoffman1,
  2. Wei Wei Le1,
  3. Luciane V. Sita2

Published Online: 1 OCT 2008

DOI: 10.1002/0471142301.ns0212s45

Current Protocols in Neuroscience

Current Protocols in Neuroscience

How to Cite

Hoffman, G. E., Le, W. W. and Sita, L. V. 2008. The Importance of Titrating Antibodies for Immunocytochemical Methods. Current Protocols in Neuroscience. 45:2.12:2.12.1–2.12.26.

Author Information

  1. 1

    Department of Anatomy and Neurobiology, University of Maryland, Baltimore, Maryland

  2. 2

    Department of Anatomy, University of São Paulo, São Paulo, Brazil

Publication History

  1. Published Online: 1 OCT 2008
  2. Published Print: OCT 2008

This is not the most recent version of the article. View current version (1 JUL 2016)


When using immunocytochemistry, investigators may not know how to optimize staining or how to troubleshoot the method when staining fails. Lacking are guides for comparing techniques and applying information derived from one staining method to another. Newer methods amplify signal detection, but will not necessarily work at the same primary antibody concentrations used for less sensitive reactions. Recommendations of optimal titers are often not accurate and are not usually accompanied by information on the method used to test those antibodies or the specifics of the assay. When the staining does not work, the investigators do not know how to determine if the antiserum is bad, the tissue is bad, or the method is inappropriate for their staining. This unit describes detailed procedures for determining optimal staining and applying that information to three common immunofluorescence methods. Lastly, a formula is provided for converting among the different methods. Curr. Protoc. Neurosci. 45:2.12.1-2.12.26. © 2008 by John Wiley & Sons, Inc.


  • immunoperoxidase;
  • ABC technique;
  • TSA amplification;
  • immunofluorescence;
  • immunocytochemistry;
  • antibody dilution