BIOTECH 304

IMMUNOHISTO CHEMISTRY

Immunohistochemistry (IHC) is the most common

application of immunostaining. It involves the process of
selectively identifying antigens (proteins) in cells of a
tissue section by exploiting the principle of antibodies
binding specifically to antigens in biological tissues.

IHC has existed since the 1930s, but it was not until 1941 that
the first IHC study was reported. Coons and his colleagues
used Fluorescein isothiocyanate (FITC)- labeled antibodies
with a fluorescent dye to localize pneumococcal antigens in
infected tissues. With the expansion and development of IHC
technique, enzyme labels have been introduced, such as
peroxidase and alkaline phosphatase. Colloidal gold label has
also been discovered and used to identify
immunohistochemical reactions at both light and electron
microscopy levels. Other labels include radioactive elements,
and the immunoreaction can be visualized by
autoradiography. The aim of IHC is to perform most IHC
staining by causing least damage on the cell or tissue, and by
using least amount of antibody, it finds a way in the tumor
typing and tumor markers.
The principle of IHC has existed since the 1930s, but it was not until 1941 that the first IHC
study was a
The IHC technique was invented during the 1940s
(Coons, Creech, & Jones, 1941) and is routinely used as
an important tool in health care and pathology for e.g.
diagnostic purposes or to stratify patients for optimized
treatment regimes. IHC is also widely used in research
where molecules of interest are analyzed to study their
roles in both healthy and diseased cells and tissues on
the molecular, cellular or tissue level. There are many
different ways to perform visualization of targets in
tissues using IHC or IHC-based methods, and numerous
protocols exist for different applications and assays.
Even though IHC is generally a robust and established
method, new assays often need careful optimization
depending on the tissue or on the properties of the
target protein, binder-molecule and/or reporter system.
Many years of technical development and the hugely
increased availability for specific binding-molecules have
greatly improved the usefulness and areas of
applications for IHC. The progress in the field of IHC-
based techniques and reagents has enabled scientists
and health care providers with more precise tools,
assays and biomarkers. In addition, technical advances
have enabled e.g. highly sensitive simultaneous
detection of multiple proteins in the same sample, and
the detection of protein-protein interactions.

The classical IHC assay involves detection of epitopes

expressed by a single protein-target within a tissue
sample using a "primary antibody" capable of binding
those epitopes with high specificity. After the epitope-
antibody binding event, a "secondary antibody" capable
of binding the primary antibody with high specificity is
added. The secondary antibody is coupled to a reporter
molecule and after the antibody-antibody binding event,
a chemical substrate is added which reacts with the
reporter molecule to produce a colored precipitate at the
site of the whole epitope-antibody complex.

The basic principle of immunohistochemistry.

a formalin-fixed paraffin embedded tissue section is
stained using a primary antibody directed towards a
specific protein target. A solution containing the primary
antibody is added to the tissue section and the
antibodies are allowed some time to find and bind to
their target. After this step, unbound and surplus
antibodies are washed away and the secondary
antibody is added. The secondary antibody, which
carries a linker molecule with horseradish peroxidase
(HRP) enzymes, is also allowed some time to bind to the
primary antibody, followed by another washing step.
After this, 3,3' Diaminobenzidine (DAB) is added. The
HRP enzyme transforms the DAB substrate into a
brownish precipitate that is deposited in the tissue at the
site of the reaction, thus producing a visual
representation of where the primary antibody first bound
its target.
USES
IHC is widely used in both research and clinical practice.
The Human Protein Atlas (HPA) project is a prime
example of how high-throughput IHC is used to achieve
large-scale mapping of the human proteome in a
multitude of tissues, cancers and cells. In the HPA
project, a streamlined in-house large scale antibody
production chain facilitates the generation of specific
antibodies, which after passing basic characterization
and validation regimes, are used to systematically stain
tissue microarrays containing hundreds of tissue cores
within a single experiment. The system for IHC
employed by HPA relies heavily on standardization of
protocols and automatisation using machines, but the
evaluation of the optimal titration for each antibody is
performed manually before the antibody is approved for
staining on the full set of tissues. Each stained tissue
core is annotated with respect to immunohistochemical
staining in tissues and cell types, and thereafter
published as a high resolution image on the web portal
to be freely viewed by anyone.
In clinical practice, IHC is mainly used within pathology
to aid physicians to evaluate tissue specimens with
respect to healthy and or diseased states, to set
diagnoses, and to define the molecular subtype of
different types of cancer. A specific example where IHC
is used diagnostically is when pathologists are
presented with a metastatic tumor sample and the tissue
origin of the primary tumor is unknown. In these cases,
pathologists use a panel of different antibodies that
target tissue specific proteins, such as prostate-specific
antigen for prostate cancer, or estrogen receptor for
gynecological cancers, or cytokeratin 20 for
gastrointestinal cancers. Once a broad classification is
made, additional tissue specific antibodies are used to
further pinpoint the origin of the primary tumor. This
information is useful for choosing the best or most
appropriate strategy for drug therapy and/or to locate the
primary tumor for radiation therapy and surgery.
Types of Staining
Immunostaining can be performed either directly or
indirectly. Conjugating our application-validated
antibodies to a number of fluorophores enables direct
labeling. To browse our offering of direct conjugates,
Often based on antigen expression levels and
accessibility, a direct conjugate is not sufficient to
visualize the target of interest. In these cases, indirect
labeling is employed using a secondary antibody. The
preferred method at CST is to utilize highly sensitive,
one-step, polymer-based detection reagents specific for
rabbit or mouse IgG.
THANKING YOU
SUBMITTED BY: DIVNOOR
GARCHA
SUBMITTED TO : DR. RATAN
CHOUDHARY