Immunohistochemistry
Irfan Zafar
MPhill Microbiology
Immunohistochemistry
PRINCIPLE OF IMMUNOHISTOCHEMISTRY (IHC)
Immunohistochemistry (IHC), or immunohistochemical
staining, is a technique which employs antibodies to detect
antigens in cells within a tissue section. This application is
used to locate specific antigens in tissue sections with
labeled antibodies based on antigen-antibody interactions
Immunohistochemistry
• Advancements in protein conjugation, tissue fixation
methods, detection labels and microscopy have allowed
Immunohistochemistry to become an essential tool in
diagnostic and research laboratories.
Immunohistochemistry is commonly used by clinicians to detect and
diagnose abnormal cells found in disease states such as cancer. Such
biomarkers are specific to the disease state and are characteristic of
particular events such as cell death, apoptosis or proliferation, which
give rise to the abnormality
Immunohistochemistry
Immunohistochemistry can also be used:
1. As a predictor for treatment, prognosis and outcome
2. During basic research, to evaluate the location and co
localization of proteins within a cell, for instance in the
nucleus, cytoplasm or membrane
Immunohistochemistry
Fixation
The principle of IHC tissue fixation is to maintain tissue
structure and retain antigenicity. Users must be sure to
preserve the readable tissue architecture and cell
morphology, otherwise the localization of immune reactive
products cannot be recognized. However, if the tissue is
over-fixed, the antigenicity will be diminished or even
completely extinguished thus resulting in false negative
staining.
Immunohistochemistry
Antigen retrival
After fixation, the epitopes may be cross-linked and
covered making it difficult for antibody-binding. By pre-
treatment with antigen retrieval reagents or procedures,
investigators can re-open the cross-linked epitopes so that
antibodies can easily bind to target antigens. Several
approaches to antigen retrieval have been widely
published, including heat and enzyme retrieval.
Immunohistochemistry
Methods
Blocking Several endogenous substances may interfere
with IHC results, such as endogenous peroxidase,
fluorescence, antibody binding capability or biotin.
Therefore, blocking the endogenous material prior to
staining is crucial in IHC to avoid false positive staining
Immunohistochemistry
Methods
• Visualization of the Antibody-Antigen Complex There are
several potential options available to visualize the antibody-
antigen complex in immunohistochemistry:
• 1. By directly conjugating a fluorophore such as rhodamine or
fluorescein
• 2. Indirectly through the use of a secondary antibody conjugated
either to an enzyme or a fluorophore
Immunohistochemistry
Methods
• The specificity of an antibody refers to its ability to recognize a
specific epitope in the presence of other epitopes. The use of an
antibody with high specificity will result in less cross-reactivity.
With respect to native protein antigens, the binding affinity of
labeeled antibody is more
Immunohistochemistry
Methods
• The measure of the binding strength of an antibody for a
monovalent epitope is referred to as affinity. Affinity can only be
estimated with pAbs because they are composed of numerous
antibodies of varying affinities. Antibodies with high affinity
bind larger amounts of antigen with a greater stability in a shorter
time than those with low affinity and are preferable for
immunohistochemical techniques
Immunohistochemistry
Direct and Indirect Methods
Despite the potential advantages of direct detection, many
immunoassays today still employ the principle of indirect
detection. Undoubtedly the main reason for this is that direct
labeling of primary antibodies is relatively complicated and
indeed, historically, antibody labeling has been carried out only
by those with specialist knowledge of chemical-modification
techniques.
Immunohistochemistry
Direct and Indirect Methods
• With indirect detection, the label is covalently attached to a
secondary antibody, which is allowed to bind to the primary
antibody in the immunoassay. Alternatively, using direct
detection, the label is attached via a covalent bond to the primary
antibody
The complexity of
Immunohistochemistry
It is an expensive task for the commercial market to provide all
antibodies pre-labeled. Despite this, scientists still need access to
such conjugates to advance their scientific research and so it is
necessary for the end user to label commercially sourced
antibodies in a cost effective manner whilst simultaneously
overcoming the difficulties of conventional labeling approaches
such as antibody loss.
Immunohistochemistry
Counterstains
• After immunohistochemical staining of the target antigen, a
second stain is often applied to provide contrast that helps the
primary stain stand out. Many of these stains show specificity
for discrete cellular compartments or antigens, while others will
stain the whole cell.
Immunohistochemistry
Advantages
• It is possible to use fresh or frozen tissue samples for IHC.
• IHC is well-established and readily available.
• It has a fast turn-around time.
• Because no live infectious agents are involved, the risk to human
health is minimal.
•
Immunohistochemistry
Disadvantages
• IHC stains are not standardized worldwide.
• While the cost of the procedure is relatively inexpensive, the
equipment needed to perform IHC is costly.
• Quantifying results is difficult.
• IHC is subject to human error. Well-trained personnel are paramount.
