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Immunohistochemistry

Florence M Hofman1, Clive R Taylor1

  • 1University of Southern California, Los Angeles, California.

Current Protocols in Immunology
|February 11, 2014
PubMed

Insights

This guide details immunohistochemistry (IHC) methods for antigen localization in tissues and cells. It covers sample preparation, multi-antigen detection, and troubleshooting techniques like antigen retrieval and preabsorption for accurate results.

Area of Science:

  • Biotechnology
  • Cell Biology
  • Histology

Background:

  • Immunohistochemistry (IHC) is a powerful technique for visualizing antigen localization within biological samples.
  • Standard IHC protocols require careful optimization for different tissue types and antigen sensitivities.

Purpose of the Study:

  • To describe various methods for antigen localization using immunohistochemistry (IHC).
  • To provide guidance on sample preparation, multi-antigen detection, and troubleshooting common IHC issues.

Main Methods:

  • Detailed protocols for preparing fresh, frozen, and fixed paraffin-embedded tissues and cells.
  • Strategies for simultaneous detection of multiple antigens.
  • Methods for antigen retrieval and secondary antibody preabsorption to enhance specificity and sensitivity.

Main Results:

  • Successful localization of specific antigens in diverse biological preparations.
  • Demonstration of simultaneous detection of two or three antigens.
  • Identification of causes for background staining and methods for its elimination.

Conclusions:

  • Multiple IHC protocols are available, each with specific advantages and disadvantages.
  • Careful selection of reagents and techniques, including antigen retrieval and preabsorption, is crucial for successful IHC.
  • Optimization of IHC methods ensures accurate antigen localization and minimizes background noise.

Keywords:
antigen retrievaldouble stainingimmunochemistryimmunohistochemistrystaining cell culturesstaining fixed tissuesstaining frozen tissues

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