Immunohistochemistry Technique

1)Immunohistochemistry (IHC) is a technique used to discern the place of

specific proteins in a tissue (1). It is done by utilizing the highly specific
interaction between an antigen and an antibody. Antibodies are produced
by immune system against antigens that are considered foreign to our
body. So in order to detect the protein we are in search for, we need an
antibody. It is carried out on formalin fixed paraffin embedded tissue.
This technical knowledge has both research and diagnostic importance and
its significance lies in:
 Classifications of neoplasm: it is used to determine the site of origin
of a metastatic tumor and accordingly classify it under the tissue
type.
 Biomarker in malignancies: it provides predictive and prognostic
information for breast, lung and gastrointestinal tract malignancies.
 Diagnose virus-infected cells: because cells in some diseases
produce proteins particular to their pathological condition.
2)Antibodies and Antigens:
Antibodies (immunoglobulins) are molecules diverse molecules that include
IgG IgM IgD IgA and IG (1). Antibodies are characterized by having high
antigen specificity. Antibody harvesting techniques: Two main methods are
used to generate antibodies which are monoclonal antibodies (mAbs) and
polyclonal antibodies (pAbs). To produce mAbs , the hybridoma technology
is used where “target antigen” sensitized B lymphocytes producing a
specific antibody are fused with myeloma cells to create immortal
hybridized cells that produce identical antibodies. However, pAbs are
created by immunizing the animal with the antigen and then the animal
produces a pool of antibodies due that recognizes different antigen epitopes.
Antibody detection techniques: Whether using primary or secondary
antibodies as will be discussed label through direct and indirect methods,
tagging the antibodies is essential. Some of the pages include ⁃
Fluorophores: commonly fluorescein isothiocyanate (FITC). Such molecules
emit vibrant signals, after being irradiated by a specific wavelength,
visualized by fluorescence microscopy. ⁃ Enzymes: this technique involves
conjugating the antibody with a specific enzyme as alkaline phosphatase
(AP). When the antibody binds to the targeted protein, its loaded enzyme
Immunohistochemistry Technique
reacts with the protein and forms a color visible reaction at the antigen
antibody site. Bright field microscopy detects it. ⁃ Metals (gold…) can also
tag antibodies
3)Sample Preparation:
To prepare the tissue for the microscopic examination, several steps
should be done, mainly including fixation, embedding, and sectioning (2).
To prevent the autolysis of the tissue, fixation is done by placing small
pieces of the tissue in a fixative solution, which is a chemical that cross-links
proteins and inactivates degradative enzymes, preserving the cell and tissue
structure. After the tissue is infiltrated with paraffin, it is placed in a mold of
melted paraffin and allowed to harden. So, the result till here is a solid block
of paraffin with the tissue imprisoned in it. Then, a microtome is used to
slice the block into thin sections. These slices are examined under the
microscope, and this is called trimming or slicing.
Antigen retrieval is the “unmasking” of an antigen epitope (3) (a
specific region on an antigen that is recognized and bound by an antibody or
immune cell receptor). There are 2 retrieval methods, Protease-induced
Epitope Retrieval (PIER), where specific enzymes are used to cleave the
peptides that may be masking the epitope, and Heat-induced Epitope
Retrieval (HIER) reverses some cross-links and allows for restoration of
secondary or tertiary structure of the epitope.
4)Immunostaining Procedure:
Now the primary antibody is applied. Primary antibodies, are antibodies that
bind directly to the epitope of a certain antigen (4), this is followed by
incubation for a certain period of time after which the sample is washed to
get rid of unbounded antibodies. And since every antigen binds only to a
certain number of primary antibodies, it would be hard to observe those
antigens clearly under a microscope, for that secondary antibodies are used.
Secondary antibodies aid to clarify the observation of antigens through
binding to primary antibodies (5). Those secondary antibodies are usually
labelled through a fluorescent dye or a specific enzyme leading to a clear
view of the antigen. It is important to note that, a control sample- one
positive and one negative- must be used to ensure that the immunostaining
process is done accurately.
Immunohistochemistry Technique
5)Detection and Visualization:
Immunohistochemistry has 2 methods of detection based on
the visualization of 2 products:
1. Chromogenic substrates focus on qualitative and quantitative visualization
of enzymes and other types of proteins by cleaving to produce a color
change that is usually picked to be high in contrast with the chosen culture.
This color will indicate that the tested enzyme is present in our culture and
was able to cleave the used substrate.
2. Fluorescent labelled antibodies that bind to the target antigen needed to be
detected. This method is usually preferred when testing the presence of 2 or
more proteins/antigens in the same culture as the signs it provides don’t
overlap or mix, making it possible to identify each antigen alone. (6)
There are 2 methods used for both chromogenic and fluorescent signal:
- direct technique where the product used is bound to a primary Ab so that
the detection is through the direct binding of the target antigen to the
antibody.
- indirect technique its more commonly used: product used is bound to a
secondary antibody, leaving the primary antibody free at the beginning. The
labelled secondary antibody would bind to the free primary antibody, which
in turn bonds to the target antigen/ protein. The extra level of antibody
binding process will amplify the signal and magnitude of detection. (7)
6) Imaging and Analysis:
Immunofluorescence (IF) is a highly sensitive technique for antigen
detection. Immuno-Ratio, a Java-based Image J plugin, incorporates various
plugins for image analysis. It was integrated into a web application using
Google Web Toolkit, Apache Commons File Upload, Apache Commons IO,
Laboratory for Optical and Computational Instrumentation Bio-Formats, and
Apache Tomcat servlet container (8). Immuno-Ratio was calibrated using 50
immunohistochemically stained slides, with a third-degree polynomial
function fitted to the data. Validation involved acquiring 12 images per
sample from 10 tumor samples using a 20× objective. Immuno-Ratio's
reliability and accuracy were evaluated. The software testing phase assessed
its robustness to staining variations and image settings. Inter-laboratory
Immunohistochemistry Technique
variability was simulated, different microscope objectives and cameras were
tested, and a first-degree polynomial was used to estimate the microscope
setup's scale. Overall, IF enables antigen detection, while Immuno-ratio
provides powerful image analysis tools. Its calibration, validation, and
testing ensure accurate results in immunohistochemistry analysis.
7)IHC Test for Cancer:
Immunohistochemistry is particularly useful in diagnosing certain types of
cancer, providing the care team with a wealth of information about the
disease to help determine treatment such as:

 Where the cancer started

 The type of cell it started in
 Whether it’s likely to grow slowly or quickly

A note ought to be mentioned here is that when the care team finds it
difficult to differentiate between cancerous and healthy cells
depending on routine tests, they tend to work on IHC. (9)

Different types of cancers can be detected by this technique, such as:

 Breast cancer:  IHC tests may detect the presence or absence

of hormone receptors on breast cancer cells. For example:
detecting the HER2 receptors indicate that this cancer is fast
growing.
 Gastrointestinal cancer:  IHC tests may help inform treatment
by determining whether H. Pylori or other factors may be
responsible for causing the cancer. It may also differentiate
between different types of it.
 Lung cancer: After other tests confirm a lung cancer diagnosis,
IHC tests may be necessary to define the type of it.
 Lymphoma: With IHC tests, the pathologist is able to test the
white blood cells causing the swelling to see whether they carry
cancer-indicating antigens

8)Advantages and Limitations:

Regarding the strength and benefits of immunohistochemistry. (10)
Immunohistochemistry Technique
 Well-established and an easy procedure that can be used with few
resources.
 Amazing technique in order to determine the presence or absence of a
target at the cellular level.
 Stained tissue samples can be stored for long period of time and
referred to whenever needed.
 Considered neither costly nor time consuming.
 Not risky to human health since no live infectious agents are being
involved.
Now moving into Limitations:
 Accuracy of antibodies can be variable and needs to be continuously
checked by other controls.
 Sample section is highly processed may lead to loss of certain
information or mistaken results.
 Highly subjected to human errors because it requires a lot of training.
 Not standardized worldwide.
9)Recent Advances in Immunohistochemistry:
We have many advances in this field such as Multiplexing and spatial
profiling immunohistochemistry allows for the simultaneous detection of
multiple targets within a single tissue section, enabling researchers to study
complex cellular interactions. (11) This technique has been further enhanced
by the development of imaging methods that enable spatial profiling,
providing information about the precise location of different cell types and
molecules within the tissue. Another important development is Quantitative
and automated analysis: Advances have been made in developing automated
systems for quantifying IHC staining, reducing the subjectivity and
variability associated with manual scoring. These systems utilize image
analysis algorithms to measure staining intensity, distribution, and other
parameters, providing more objective and reproducible results.
10)Conclusion:

Immunohistochemistry is a widely-used technique that allows for the

visualization and localization of specific proteins within tissue samples. This
technique involves the use of specific antibodies that bind to target proteins
Immunohistochemistry Technique
in the tissue. The application of immunohistochemistry has revolutionized
the field of biological research by providing a means to study protein
expression and localization in situ. Immunohistochemistry is particularly
useful in the field of pathology, as it enables the identification and
characterization of specific antigens in tissue samples. While
immunohistochemistry is a powerful tool, it does have certain limitations
that need to be considered such as the specificity of the antibodies used. It is
crucial to carefully select antibodies that specifically bind to the target
protein of interest, as non-specific binding can lead to false-positive results.

Done by:

Adam Sabra (3520) Zouheir Alawiyeh (3584) Ahmad Atieh (3569)

Riwa Al Atat (3559) Dima Zeeb (3523) Ali Dbouk (3580)

Fatima Sareeni (3593) Hadi Salloum (3507) Ibrahim Sabra (3599)

Manar Zgheib (3537) Kawthar Berro (3508) Yehya Sabra (3597)

Immunohistochemistry Technique

References:
1) 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. PMID: 30539453; PMCID:
PMC6749998.
2) Author. Mescher A.L.(Ed.), (2018). Junqueira’s Basic Histology: Text
and Atlas, 15e. McGraw Hill.
https://accessmedicine.mhmedical.com/content.aspx?
bookid=2430&sectionid=190220001
3) . www.rndsystems.com.
https://www.rndsystems.com/resources/protocols/antigen-retrieval-
methods#:~:text=Antigen%20retrieval%20refers%20to
%20any,epitope%2Dantibody%20binding%20is%20restored.
4) Primary and secondary antibodies. ProSci Incorporated. (2021, August
24). https://www.prosci-inc.com/applications-techniques/5-a-of-antibody-
development/primary-and-secondary-antibodies.
5) Introduction to secondary antibodies. Thermo Fisher Scientific - US.
(n.d.-b).
https://www.thermofisher.com/lb/en/home/life-science/antibodies/antibodies
-learning-center/antibodies-resource-library/antibody-methods/introduction-
secondary-antibodies.html
Immunohistochemistry Technique
6) https://goldbio.com/articles/article/Chromogenic-Substrates-Overview
- :~:text=Chromogenic%20substrates%20aid%20in%20the,visible%20color
%20(chromo)%20change
7) https://www.novusbio.com/ihc-detection - :~:text=Detection%20can
%20be%20performed%20through,experimental%20needs%20as
%20described%20below
8) ImmunoRatio: A publicly available web application for quantitative
image analysis of estrogen receptor (ER), progesterone receptor (PR), and
Ki-67. BioMed Central.
https://breast-cancer-research.biomedcentral.com/articles/10.1186/bcr2615
9) Immunohistochemistry: IHC Test for Cancer. City of Hope. (2022,
September 25).
https://www.cancercenter.com/diagnosing-cancer/diagnostic-procedures/
immunohistochemistry

10) Immunohistochemistry techniques, strengths, limitations and

applications. Analysis & Separations from Technology Networks.
(n.d.).
https://www.technologynetworks.com/analysis/articles/immunohistoch
emistry-techniques-strengths-limitations-and-applications-363107

11) Tan WCC, Nerurkar SN, Cai HY, Ng HHM, Wu D, Wee YTF, Lim
JCT, Yeong J, Lim TKH. Overview of multiplex
immunohistochemistry/immunofluorescence techniques in the era of
cancer immunotherapy. Cancer Commun (Lond). 2020 Apr;40(4):135-
153. doi: 10.1002/cac2.12023. Epub 2020 Apr 17. PMID: 32301585;
PMCID: PMC7170662.