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Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas
Published on: May 31, 2012
Characteristics of the tissue section that influence the staining outcome in immunohistochemistry
Sylwia Libard1,2, Dijana Cerjan2, Irina Alafuzoff3,4
1Rudbeck Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University/Uppsala University Hospital, Dag Hammarskjölds väg 20, 751 85, Uppsala, Sweden.
Insights
Section thickness significantly impacts immunohistochemistry (IHC) results, affecting protein detection in tissues. Understanding this variable is crucial for accurate diagnostic assessments and reproducible IHC staining.
Area of Science:
- Histopathology
- Biomarker Analysis
- Immunohistochemistry Techniques
Background:
- Immunohistochemistry (IHC) is a widely used technique for tissue analysis.
- Numerous pre-analytical and analytical factors can influence IHC outcomes, including tissue processing and antibody selection.
- The impact of section thickness on IHC results, particularly in complex tissues like the brain, remains underexplored.
Purpose of the Study:
- To investigate the influence of tissue section thickness on immunohistochemistry (IHC) staining.
- To highlight the importance of section thickness as a critical variable in IHC methodology.
- To emphasize the need for standardized reporting of IHC methods.
Main Methods:
- Comparative analysis of IHC staining across different section thicknesses (e.g., 4 µm vs. 7 µm).
- Assessment of protein localization and signal intensity in various tissue types.
- Consideration of antibody molecular weight and its potential interaction with section thickness.
Main Results:
- Tissue section thickness was identified as a significant factor affecting IHC outcomes.
- Variations in section thickness can lead to differential detection of cellular components and proteins.
- Thinner sections may result in the loss of detectable signals compared to thicker sections.
Conclusions:
- Section thickness is a critical, yet often overlooked, variable in immunohistochemistry.
- Standardized reporting of section thickness is essential for reproducibility and accurate interpretation of IHC results.
- Further research is needed to optimize section thickness for different antibodies and tissue types to ensure reliable biomarker assessment.
Abstract:
Immunohistochemistry (IHC) is influenced by several factors such as cold ischemia time, fixative, fixation time, paraffin, storage time, antibody, antigen retrieval technique and detection systems. In the setting of post-mortem tissue, not only post-mortem delay, but also agonal state is of interest. Here, we assessed an additional variable, i.e., the thickness of the section, and noted that this variable also influenced the IHC outcome. This is of significance when the extent of labelling is a parameter to be assessed, for example when assigning a stage or grade of a disease. Furthermore, when assessing brain tissue with neurons, soma measuring from 4 to 100 µm, various cellular compartments composed of different proteins are localised in sections measuring 4 or 7 µm. Thus, what is seen in a 7-µm-thick section might be lacking in a 4-µm-thick section. Lack of information regarding the molecular size of commercial antibodies is also disturbing as this parameter might influence the distribution of the molecule in the three-dimensional section. The choice of antibody to be used and the staining methodology have been acknowledged being of significance for IHC outcome; however, neither sections thickness or the molecular weight has been discussed sufficiently. IHC has been shown to be an unpredictable technique used for assessment of tissue. This emphasises the need for detailed methodological descriptions in publications, the need to acknowledge and to harmonize all eventual pitfalls related to this methodology.
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