Immunohistochemistry basics
Immunohistochemistry (IHC) is a widely used and versatile antibody-based technique for the detection and localization of specific antigens in cells and tissues.
It is a standard procedure in both biological research and clinical diagnostics.
Particularly in cancer pathology, it is used in classifying tumors, tracking metastases, and identifying tiny foci of new tumor cells.
IHC is applicable for a variety of tissue types, including fixed tissues, enhancing its accessibility and utility. It can also be adapted to automated workflows, enabling high-volume processing with reliable reproducibility.
Recent advances, such as multiplex immunohistochemistry, allow for the simultaneous detection of multiple markers on a single tissue section, catering to the growing demand for improved and efficient techniques.
These advancements have expanded the role of IHC in providing predictive and prognostic information, underscoring its increasing importance in both research and clinical settings.
A general IHC protocol can be broadly summarized as follows.
Tissue specimens, which can be formalin-fixed paraffin-embedded (FFPE) or fresh frozen, are sectioned into thin sections and mounted onto glass slides.
Fixed tissue will generally undergo additional processing, such as deparaffinization and antigen retrieval.
A blocking step prevents non-specific binding of antibodies to tissue antigens.
Primary antibodies specific to the target antigen are added, which can be detected directly using fluorescent or enzyme labels or indirectly with a labeled secondary antibody.
Enzyme labels require the addition of chromogenic substrates for detection.
Lastly, slides may be counterstained, mounted, and imaged under a microscope to analyze the results.
IHC for frozen sections
Fresh frozen tissue sections are often used for IHC when rapid analysis is needed, such as in surgical procedures and determining cancer margins.
Frozen samples are particularly advantageous for preserving enzymes or antigens in a more native state, which is ideal in cases where fixation might compromise immunoreactivity.
Frozen sections also provide a faster turnaround time compared to FFPE sections.
In certain cases, the staining quality can be lower partly due to the thicker size of sections typically produced, which can reduce microscopic resolution.
Ice crystals can also form during freezing, which may disrupt tissue morphology and form artifacts.
Techniques such as paraformaldehyde (PFA) perfusion fixation followed by cryopreservation in sucrose solution (known as fixed-frozen) can be used to minimize artifacts.
However, fixation with aldehyde-based reagents like formalin may require epitope retrieval to restore antigen accessibility.
IHC for fixed tissue
Formalin-fixed, paraffin-embedded (FFPE) tissue sections are a widely used sample format for IHC.
They are prepared by immersing excised tissue in formaldehyde before embedding it in paraffin wax.
This multi-step paraffinization process transitions the tissue from an aqueous to an organic phase, ensuring stable preservation.
FFPE tissues are known for their long-term storage capability even at room temperature, making them ideal for sample archiving as well as diagnostic testing.
FFPE sections offer additional advantages, including the elimination of antigen degradation due to premature thawing, better tolerance of heat-mediated antigen retrieval, and superior preservation of cellular morphology.
The solid fixation process also allows for thinner sectioning, which enhances the visualization of tissue morphology.
Careful optimization of FFPE tissue preparation is critical for achieving reliable and meaningful staining results.
Key steps include deparaffinization, antigen retrieval, permeabilization, and blocking, all of which contribute to overall staining quality.
Proper
Antigen retrieval
is particularly important, as inaccessible antigens can lead to false negative or positive results and may even cause antigen loss if tissue detaches from the slide.
Heat-induced epitope retrieval (HIER) tends to be preferred over proteolytic-induced epitope retrieval (PIER), which is harsher on tissue and best saved for heavily cross-linked samples.
Antibody selection for IHC
The accuracy and reliability of IHC results are highly dependent on selecting the appropriate antibodies, as improper selection or insufficient validation can lead to ambiguous or false findings.
Several factors must be considered to ensure optimal antibody choice for a given IHC experiment. Monoclonal antibodies, which bind to a single epitope, offer exceptional specificity and are ideal for targeting unique antigens.
In contrast, polyclonal antibodies recognize multiple epitopes, making them more sensitive but potentially less specific.
The choice of antibody label dictates the methods of detection. Enzyme labels, such as horseradish peroxidase (HRP) and alkaline phosphatase (AP), are widely favored and rely on chromogenic substrates.
Fluorescent labels, which require fluorescence microscopy, are essential for multiplexed detection and co-localization studies of multiple antigens.
Additionally, polymer-based labels can significantly enhance signal amplification, improving sensitivity for low-abundance targets. Read our
guide to IHC antibodies
to learn more.
Common stains for IHC
A variety of stains
are commonly used in immunohistochemistry for counterstaining or highlighting specific tissue structures.
Hematoxylin and eosin (H&E) are widely utilized for general tissue morphology, while specialized stains target distinct components.
These include native DNA (Methyl Green), neuronal cell bodies (Cresyl Violet), polysaccharides (Alcian Blue), lipids (Oil Red O), and iron deposits (Prussian Blue), among other stains.
Selecting the appropriate stain requires careful consideration of factors like tissue type, antigen stability, detection sensitivity, and compatibility with the primary antibody and detection system.
Researchers must also prioritize signal clarity, minimize background interference, and ensure the stain resists fading to produce high-quality, reliable images.
These considerations are essential for achieving accurate visualization and interpretation of IHC results.
Multiplex immunohistochemistry
Multiplex immunohistochemistry enables the simultaneous detection of multiple target antigens on a single tissue section.
This addresses the limitations of conventional IHC, which is generally restricted to one marker.
Multiplexing is gaining prominence in both research and clinical settings due to its ability to analyze complex tissue biology, especially when dealing with rare or limited samples.
Multiplex fluorescence-based staining technologies are ideal in the identification of specific cell subsets based on combinations of markers and their spatial relationships within tissue architecture.
High-plex immunostaining methods often face challenges such as low throughput, time-consuming development, and constraints on primary antibody availability due to species or isotype cross-reactivity.
Recent advancements, like hapten-based labeling systems, offer scalable and efficient chromogenic or fluorescent multiplex detection, simplifying antibody selection and improving accessibility.
Other emerging techniques, including cyclic immunofluorescence and tyramide-based mIHC/IF, are expanding capabilities to achieve higher sensitivity, greater plexing, and more detailed spatial resolution.
Digital immunohistochemistry
Digital IHC is a branch of digital pathology that focuses on IHC-stained slides.
By capturing high-resolution images of entire stained tissue sections, digital IHC enables automated analysis using specialized software.
Using digital slide scanning methods, researchers and clinicians gain instant access to digital slides, eliminating the need to transport fragile glass slides between locations.
This not only reduces the risk of damage but also minimizes human error and subjectivity in data interpretation.
Additionally, it allows for a shift from semi-quantitative assessments to objective, fully quantifiable results.
Digital IHC also enhances data sharing and education by creating a centralized repository of pathological cases, including rare samples.
It simplifies the reanalysis of markers over time and is far more effective at managing multiplexed data compared to traditional methods.
Researchers can efficiently study multiple markers in parallel and detect subtle variations in staining intensity, leading to deeper insights into their samples.
Key considerations when adopting digital IHC include choosing between brightfield or fluorescence-based imaging, ensuring the necessary IT infrastructure to support a digital platform, and integrating slide scanners with automated processing systems.
Read an
introduction to digital IHC here.
References
Magaki S, Hojat SA, Wei B, So A, Yong WH. An Introduction to the Performance of Immunohistochemistry. Methods Mol Biol. 2019;1897:289-298. doi:10.1007/978-1-4939-8935-5_25
Mason, E. Tips for Successful IHC Staining. Biocompare. 2021 May 11 [cited 2024 Dec]. Available from: www.biocompare.com/Editorial-Articles/574807-Tips-for-Successful-IHC-Staining/
Mason, E. Avoiding Common Immunohistochemistry Mistakes. Biocompare. 2023 Apr 20 [cited 2024 Dec]. Available from: www.biocompare.com/Editorial-Articles/595864-Avoiding-Common-Immunohistochemistry-Mistakes/
Kent-Webb, H. Immunohistochemistry vs Immunocytochemistry. Biocompare. 2024 Dec 5 [cited 2024 Dec]. Available from: www.biocompare.com/Editorial-Articles/615999-Immunohistochemistry-and-Immunocytochemistry/
Tan WCC, Nerurkar SN, Cai HY, et al. Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy. Cancer Commun (Lond). 2020;40(4):135-153. doi:10.1002/cac2.12023
Mason, E. Optimizing Your Immunohistochemistry Workflow. 2020 Jan 30 [cited 2024 Dec]. Available from: www.biocompare.com/Editorial-Articles/558961-Optimizing-Your-Immunohistochemistry-Workflow/
Mason, E. Sample Preparation for FFPE Immunohistochemistry. 2019 Feb 19 [cited 2024 Dec]. Available from: www.biocompare.com/Editorial-Articles/357614-Sample-Preparation-for-FFPE-Immunohistochemistry/
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