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LEARN : METHODS > IMMUNOHISTOCHEMISTRY

Immunohistochemistry

Immunohistochemistry (IHC) is a powerful microscopy-based technique for visualizing cellular


proteins or other macromolecules in tissue samples. The strength of IHC is the intuitive visual
existence and localization of the target-protein in the context of different cell types, biological s
localization within complex tissues.

The IHC technique was invented during the 1940s (Coons, Creech, & Jones, 1941) and is rou
tool in health care and pathology for e.g. diagnostic purposes or to stratify patients for optimize
also widely used in research where molecules of interest are analyzed to study their roles in b
cells and tissues on the molecular, cellular or tissue level. There are many different ways to pe
in tissues using IHC or IHC-based methods, and numerous protocols exist for different applica
though IHC is generally a robust and established method, new assays often need careful optim
tissue or on the properties of the target protein, binder-molecule and/or reporter system. Many
development and the hugely increased availability for specific binding-molecules have greatly
and areas of applications for IHC. The progress in the field of IHC-based techniques and reag
and health care providers with more precise tools, assays and biomarkers. In addition, technic
Immunohistochemistry e.g. highly sensitive simultaneous detection of multiple proteins in the same sample, and the d
Technology interactions (see Proximity Ligation Assay).

Specific examples The classical IHC assay is illustrated in Figure 1 and involves detection of epitopes expressed
References and Links within a tissue sample using a "primary antibody" capable of binding those epitopes with high
antibody binding event, a "secondary antibody" capable of binding the primary antibody with h
secondary antibody is coupled to a reporter molecule and after the antibody-antibody binding e
is added which reacts with the reporter molecule to produce a colored precipitate at the site of
complex.

Figure 1. The basic principle of immunohistochemistry.

In the schematic illustration (Figure 1) a formalin-fixed paraffin embedded tissue section is sta
antibody directed towards a specific protein target. A solution containing the primary antibody
section and the antibodies are allowed some time to find and bind to their target. After this ste
antibodies are washed away and the secondary antibody is added. The secondary antibody, w
molecule with horseradish peroxidase (HRP) enzymes, is also allowed some time to bind to th
by another washing step. After this, 3,3' Diaminobenzidine (DAB) is added. The HRP enzyme
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substrate into a brownish
information. precipitate
Don't that
show this is deposited in the tissue at the site of the reaction, th
again.
representation of where the primary antibody first bound its target
representation of where the primary antibody first bound its target.

Technology

MENU HELP NEWSTissue preparation


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The tissue plays a central role in the experiment and it is important that it is processed so that
morphology is preserved. The most common processing for IHC is to prepare formalin-fixed pa
tissue blocks. The purpose of formalin fixation is to produce chemical cross-linking of proteins
terminates all cellular processes and freezes the cellular components at the place and in the c
the time of fixation and also prevent degradation. After adequate fixation, the tissue is further p
embedded in paraffin blocks, which are then sectioned into thin slices (usually 4-10µm) using
are transferred to glass slides and allowed to adhere prior to further processing.

Other methods for fixation besides formalin are sometimes used. These include other types of
alcohol solutions. The best choice of fixative is very much dependent on the assay. A common
prepare frozen tissue samples. In this case, the tissue is embedded in a cryoprotective medium
performed post-sectioning. Frozen tissues are sectioned in cryostats and have the advantage
and of better preservation of sensitive epitopes, but can often be inferior to FFPE tissues in te
histological morphology.

Antigen (epitope) retrieval

A concern associated with cross-linking fixatives like formalin, or too long time spent in fixative
epitopes, which can obstruct the primary antibody from binding to its target. Especially with FF
need to revert some of the chemical crosslinking and "retrieve" the epitopes before proceeding
are several antigen retrieval protocols available and the main strategies include treating the tis
digestive enzymes, detergents, or combinations thereof. The most common method for antige
is to pressure-boil the tissue slides in an acidic citrate buffer for around 15-20 minutes.

Antibody binding

The quality and specificity of the binding molecule is crucial for any IHC based technique, and
directly affect the outcome, reliability, and possibly also the interpretation of the assay. Antibod
common type of binding-molecule used for IHC, and although most antibodies are able to ade
molecule of interest, they may also vary greatly in their specificity for their intended target. Ant
are therefore more reliable when interpreting "on-target" binding, since they produce little or no
"background". Antibodies that are less specific can produce more off-target binding, and the re
possibly interfere with the correct interpretation of the true on-target signals. There are two ma
polyclonal antibodies which is a heterogeneous mix of antibodies that bind different epitopes o
antibodies that all bind the same epitope. Polyclonal antibodies are often very potent due to th
multiple epitopes on the same target. However, the epitopes they bind are often poorly defined
varying epitope-specificities comes the increased likelihood of off-target binding events and ba
the potency of polyclonal antibodies can be advantageous since the concentration of binding e
molecule usually outweighs potential background noise. A drawback is that polyclonal antibod
resources since they are derived from animal sera. Monoclonal antibodies, by contrast, have m
can be produced in hybridoma cell lines. Monoclonal antibodies are also often well defined in t
can still generate results that are hard to interpret if the specificity is low or if the target epitope
abundance.

Careful optimization and titration of antibody concentration for each assay is needed, since the
only on the antibody's specificity and affinity for the target, but also on the concentration and a
potential off-target epitopes present in the sample. Adding too much antibodies to the sample
possible low-affinity off-target binding events once the on-target epitope(s) are saturated with b
antibody concentration, off-target binding events become rarer as they usually have lower affin
events. The risk when attempting to reduce background while using a low-affinity antibody is th
concomitantly weakened to the point of providing a false negative result.

Other types of binder molecules sometimes used in IHC-based techniques include affibodies,
fragments
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Detection systems
Detection systems

The whole purpose of performing IHC is to obtain a visual representation of where the target c
experimental tissue, and preferably also gain information about the target's expression pattern
MENU HELP NEWSpopulations and/or subcellular localizations. This is exemplified in Figure 2, which illustrates ho
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used to visualize different cellular or tissue compartments within a complex tissue. To visualize
interaction, some kind of detection system that produces an observable stain or signal is need
method for introducing a detection system to the experiment is to use a secondary antibody th
reporter molecule, i.e. enzyme or fluorophore. Secondary antibodies are usually targeted spec
molecules from a different animal species. For example, if the primary antibody is raised in a r
antibody must be raised in another animal and targeted specifically towards rabbit antibodies.

Esophagus Magnification

Hematoxylin staining No antibo

p63
Nuclea
CAB000083

EGFR
Membran
CAB000035

G6PD
Cytoplasm
HPA000247

LAMB2 (Laminin)
Connec
CAB000053

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Visualizing continue,
different we'lltargets
protein assume that you are
in complex happy
tissues. to right
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ma
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corresponding images in the left column.
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In the IHC image (Figure 2), consecutive sections of human esophagus stained using four diffe
direct comparison of different protein expression patterns within the tissue and within subcellu
images are only counterstained for hematoxylin for comparison. The p63 antibody stains cell n
that reside in the basal part of the esophageal epithelium. The EGFR (Epidermal growth facto
to stain the same cell population as p63, but stains cellular membranes instead of nuclei. The
phosphate dehydrogenase) antibody stains the cytoplasm of a wider repertoire of esophageal
residing in the connective tissue. The Laminin (LAMB2) antibody stains only cells and structur
underlying the esophagus.

For FFPE tissue samples the most common detection method is to use enzymatic reactions to
precipitate at the site of antibody binding. The secondary antibodies then carry an enzyme, e.g
(HRP) or alkaline phosphatase (AP), that are capable of converting chromogens like 3,3' Diam
bromo-4-chloro-3-indolyl phosphate/ p-nitroblue tetrazolium chloride (BCIP/NBT) into brown o
deposited in the tissue at the site of the reaction. Chromogenic stains are observable in light-m
very stable over long periods of time, which is beneficial if the experiment needs to be archive
point.

For frozen tissue sections it is more common to use fluorophore-linked secondary antibodies t
(usually green, red, or blue) when excited by the correct wavelengths of light. Moreover, fluoro
stable for long periods of time. However, the benefit of using fluorophores is that they provide
performing double-labeling experiments where several antibodies towards multiple targets are
sample. The secondary antibodies need to be targeted towards different primary antibodies an
different fluorophores. The different secondary antibodies are then observed separately by exc
different wavelengths of light. These different excitation results are saved as separate images
later be overlaid to infer protein co-localizations etc.

Using reporter-carrying secondary antibodies for detection is in itself an amplification step sinc
antibodies are able to bind a single primary antibody, but sometimes further amplification steps
signal and sensitivity of the experiment. In such cases, the secondary antibody may instead ca
instance biotin polymers, which are able to recruit a larger number of reporter molecules in su
strategy for amplifying signals is useful for both enzymatic and fluorescent detection methods.

Counterstaining

Immunohistochemical staining using chromogens often benefits from having a counterstain ap


contrast and facilitates the observation of histological features. The most common type of cou
samples is hematoxylin that stains cellular cytoplasm with a pale bluish color, and stain cell nu
nuance. Fluorescent stainings are usually not counterstained with hematoxylin, since the dete
light microscopy. Instead, the most common way to obtain counterstaining for fluorescence is t
fluorescent dyes that bind nucleic acids. After the actual immunohistochemical reaction, the on
coverslip and seal the sample for protection and longterm storage. The most common way is t
sample using commercially available purpose-made resins.

Specific examples

IHC is widely used in both research and clinical practice. The Human Protein Atlas (HPA) proje
how high-throughput IHC is used to achieve large-scale mapping of the human proteome in a
and cells. In the HPA project, a streamlined in-house large scale antibody production chain fac
specific antibodies, which after passing basic characterization and validation regimes, are use
tissue microarrays containing hundreds of tissue cores within a single experiment. The system
relies heavily on standardization of protocols and automatisation using machines, but the eval
for each antibody is performed manually before the antibody is approved for staining on the fu
stained tissue core is annotated with respect to immunohistochemical staining in tissues and c
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published as a high resolution
information. image
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the web portal to be freely viewed by anyone.
In clinical practice, IHC is mainly used within pathology to aid physicians to evaluate tissue spe
healthy and or diseased states, to set diagnoses, and to define the molecular subtype of differ
specific example where IHC is used diagnostically is when pathologists are presented with a m
MENU HELP NEWSthe tissue origin of the primary tumor is unknown. In these cases, pathologists use a panel of d
LEARN : METHODS > IMMUNOHISTOCHEMISTRY
target tissue specific proteins, such as prostate-specific antigen for prostate cancer, or estroge
cancers, or cytokeratin 20 for gastrointestinal cancers (Gremel et al., 2014). Once a broad clas
additional tissue specific antibodies are used to further pinpoint the origin of the primary tumor
for choosing the best or most appropriate strategy for drug therapy and/or to locate the primar
and/or surgery.

References and Links

Original reference describing the IHC technique:


Coons, A. H., Creech, H. J., & Jones, R. N. (1941). Immunological Properties of an An
Fluorescent Group.
Experimental Biology and Medicine, 47(2), 200-202. DOI:10.3181/00379727-47-13084

Commonly used antibodies for cancer diagnostics:


Gremel, G., Bergman, J., Djureinovic, D., Edqvist, P.-H., Maindad, V., Bharambe, B. M
systematic analysis of commonly used antibodies in cancer diagnostics.
Histopathology, 64(2), 293-305. DOI: 10.1111/his.12255. PubMed: 24330150

A review on validating antibodies for IHC:


O'Hurley, G., Sjöstedt, E., Rahman, A., Li, B., Kampf, C., Pontén, F., ... Lindskog, C. (2
out: a critical evaluation of strategies used for validation of immunohistochemical biom
Molecular Oncology, 8(4), 783-98. DOI: 10.1016/j.molonc.2014.03.008. PubMed: 2472

"The Immunohistochemical Staining Methods Education Guide" - a free handbook prov


http://www.agilent.com/cs/library/technicaloverviews/public/08002_ihc_staining_metho

Antibodypedia - An open-access database of publicly available antibodies and their us


applications:
http://www.antibodypedia.com

IHC world - Protocols, Forum, Products, and more:


http://www.ihcworld.com/

A video-clip describing tissue microarrays, IHC, and the Protein Atlas website:
http://www.jove.com/video/3620/production-tissue-microarrays-immunohistochemistry-

A YouTube clip illustrating IHC from BioGenexLaboratories:


http://www.youtube.com/watch?v=HdBgTAAi3rU

Contact The Project The Human Protein Atlas


NEWS ARTICLES INTRODUCTION ANTIBODY AVAILABILITY
PRESS ROOM ORGANIZATION LICENCE & CITATION
PUBLICATIONS HELP & FAQ
DOWNLOADABLE DATA
contact@proteinatlas.org

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