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Description: Immunohistochemistry (IHC) is a technique routinely used in Anatomic Pathology to identify tissue and cellular components by using antigen-antibody reactions. Identifying these different components can assist the pathologist in diagnosing tissue/cellular abnormality or malignancy and/or etiology. Learn the basic principles and techniques for performing immunohistochemistry and immunofluorescent stain methods. You will examine an overview of various IHC techniques, and discover uses, reagents, precautions and limitations.

Start Date: Upon registration

Completion: Up to 52 weeks

Prerequisite/Corequisite Learning:

• Discuss/define terminology and basic principles of Ag-Ab reactions
• Discuss/define terminology and basic principles of enzyme reactions
• Describe the immune system and its cells/tissue/organs
• Recognize Hematoxylin and Eosin (H&E) stained normal structure of the immune system using light microscope
• Recognize H&E stained normal tissue structure using light microscope
• Describe the structure of antibodies and types of antibodies
• Define antigenic determinants
• Distinguish between monoclonal and polyclonal antibodies
• Describe basic principles of tissue/cell fixation and preservation; tissue processing and microtomy
• Describe basic theories of stains and dyes

Learning Outcomes:

• Identify the use and purpose for performing immunohistochemistry staining
• Define/describe the immunohistochemistry principles and mechanisms underlying Ag- Ab reactions:
  • Single step methods
  • Multiple step methods
    • Peroxidase anti-peroxidase (PAP) method
    • Avidin-biotin (ABC) and labelled streptavidin biotin (LSAB) methods
    • Polymer method
• Identify enzyme reactions in immunohistochemistry
• Discuss and compare visualization and amplification methods:
  • Identify use of Fluorescent dyes
  • Identify use of chromogenic dyes-Enzyme/Chromogen systems:
  • Peroxidase and DAB (diaminobenzidine) or AEC (aminoethylcarbazole)
  • Phosphatase and fast red
• Describe the limitations of IHC staining:
  • Define and describe antigen preservation
  • Recognize and describe antigen localization
  • Discuss optimal fixation methods
• Identify antigen specificity
  • Describe non-specific/specific background staining and potential causes
  • Identify use of blocking methods to increase Ag-Ab binding specificity
• Identify antibody availability and differentiate between:
  • Monoclonal antibodies
  • Polyclonal antibodies

Author: Masaye Tanaka, ART, BSc, MEd

Version Date: September 2019

PEP hours: 16.5

CPS credits: 1.6

*Note: PEP hours and/or CPS credits will only be awarded upon successful completion of Final Exam.