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Immunohistochemistry

Florence Hofman1

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

Current Protocols in Immunology
|April 25, 2008
PubMed

Insights

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

Area of Science:

  • Biotechnology
  • Cell Biology
  • Histology

Background:

  • Immunohistochemistry (IHC) is crucial for visualizing antigen distribution in biological samples.
  • Standard IHC protocols require optimization for different tissue types and antigen sensitivities.
  • Challenges include sample preparation, antigen accessibility, and non-specific antibody binding.

Purpose of the Study:

  • To describe various immunohistochemistry (IHC) methods for antigen localization.
  • To provide protocols for preparing diverse biological specimens for IHC analysis.
  • To offer solutions for common IHC challenges, including antigen unmasking and background noise reduction.

Main Methods:

  • Preparation of fresh, frozen, and paraffin-embedded tissue sections.
  • Isolation and culture of unfixed cells for IHC.
  • Application of antigen retrieval techniques for fixative-sensitive antigens.
  • Preabsorption of secondary antibodies to minimize cross-reactivity.
  • Simultaneous detection of multiple antigens using optimized reagent selection.

Main Results:

  • Detailed protocols for preparing various tissue and cell samples suitable for IHC.
  • Demonstration of simultaneous two-antigen detection capabilities.
  • Explanation of antigen retrieval and antibody preabsorption as effective troubleshooting strategies.
  • A comparative table summarizing advantages and disadvantages of different IHC approaches.
  • Guidance on identifying and eliminating background staining in IHC.

Conclusions:

  • Effective antigen localization in diverse biological samples is achievable through optimized IHC protocols.
  • Techniques like antigen retrieval and antibody preabsorption enhance IHC specificity and sensitivity.
  • Careful protocol selection and troubleshooting are essential for reliable IHC results.

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