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Optimization, Design and Avoiding Pitfalls in Manual Multiplex Fluorescent Immunohistochemistry
Published on: July 26, 2019
A Dermatopathologist's Guide to Troubleshooting Immunohistochemistry Part 1: Methods and Pitfalls
Amanda F Marsch1, Jonathan N Truong, Melissa M McPherson
1*Dermatology Resident, Department of Dermatology, University of Illinois at Chicago, Chicago, IL; and †Laboratory Manager (J.N.T.), IHC HistoTechnician (M.M.M.), Dermatopathologist (J.M.J.-H.), Director (D.M.E.), Ackerman Academy of Dermatopathology, University of Illinois, Chicago, IL.
Immunohistochemistry (IHC) techniques detect specific antigens in tissue using antibodies and enzymatic reactions. This review details IHC methodologies, common issues, and solutions for accurate diagnostic staining.
Area of Science:
- Histopathology and Diagnostic Pathology
- Molecular Biology Techniques
- Biomedical Research
Background:
- Immunohistochemistry (IHC) is a crucial technique for detecting specific antigens in formalin-fixed paraffin-embedded tissues.
- It relies on antibody-antigen interactions visualized through enzymatic reactions.
- Epitope masking during fixation necessitates retrieval methods like heat-induced or proteolytic-induced epitope retrieval.
Purpose of the Study:
- To provide a comprehensive review of immunohistochemistry (IHC) methodologies.
- To identify and explain common problems and artifacts encountered in IHC staining.
- To offer solutions for resolving technical challenges and improving diagnostic accuracy.
Main Methods:
- Detailed explanation of various IHC methodologies, including antibody conjugation and detection systems (e.g., biotin-avidin, biotin-streptavidin).
- Discussion of epitope retrieval techniques: heat-induced epitope retrieval (HIER) and proteolytic-induced epitope retrieval (PIER).
- Analysis of common staining issues and artifacts, such as non-specific staining, background noise, and weak signal.
Main Results:
- Identification of specific challenges associated with different IHC protocols and reagents.
- Elucidation of the causes behind common IHC artifacts, including fixation, processing, and antibody issues.
- Presentation of practical strategies for troubleshooting and optimizing IHC staining.
Conclusions:
- Standardized IHC protocols and careful optimization are essential for reliable results.
- Understanding potential pitfalls and artifact causes allows for effective problem-solving.
- This review serves as a guide for researchers and diagnosticians to enhance IHC assay performance.
