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Immunohistochemistry (IHC) is the most common application of immunostaining. Immunohistochemistry_sentence_0

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. Immunohistochemistry_sentence_1

IHC takes its name from the roots "immuno", in reference to antibodies used in the procedure, and "histo", meaning tissue (compare to immunocytochemistry). Immunohistochemistry_sentence_2

Albert Coons conceptualized and first implemented the procedure in 1941. Immunohistochemistry_sentence_3

Visualising an antibody-antigen interaction can be accomplished in a number of ways, mainly either of the following: Immunohistochemistry_sentence_4


Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. Immunohistochemistry_sentence_5

Specific molecular markers are characteristic of particular cellular events such as proliferation or cell death (apoptosis). Immunohistochemistry_sentence_6

Immunohistochemistry is also widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue. Immunohistochemistry_sentence_7

Sample preparation Immunohistochemistry_section_0

Preparation of the sample is critical to maintain cell morphology, tissue architecture and the antigenicity of target epitopes. Immunohistochemistry_sentence_8

This requires proper tissue collection, fixation and sectioning. Immunohistochemistry_sentence_9

A solution of formalin is often used to fix tissue, but other methods may be used. Immunohistochemistry_sentence_10

Preparing tissue slices Immunohistochemistry_section_1

The tissue may then be sliced or used whole, dependent upon the purpose of the experiment or the tissue itself. Immunohistochemistry_sentence_11

Before sectioning, the tissue sample may be embedded in a medium, like paraffin wax or cryomedia. Immunohistochemistry_sentence_12

Sections can be sliced on a variety of instruments, most commonly a microtome, cryostat, or vibratome. Immunohistochemistry_sentence_13

Specimens are typically sliced at a range of 3 µm-5 μm. Immunohistochemistry_sentence_14

The slices are then mounted on slides, dehydrated using alcohol washes of increasing concentrations (e.g., 50%, 75%, 90%, 95%, 100%), and cleared using a detergent like xylene before being imaged under a microscope. Immunohistochemistry_sentence_15

Depending on the method of fixation and tissue preservation, the sample may require additional steps to make the epitopes available for antibody binding, including deparaffinization and antigen retrieval. Immunohistochemistry_sentence_16

For formalin-fixed paraffin-embedded tissues, antigen-retrieval is often necessary, and involves pre-treating the sections with heat or protease. Immunohistochemistry_sentence_17

These steps may make the difference between the target antigens staining or not staining. Immunohistochemistry_sentence_18

Reducing non-specific immuno-staining Immunohistochemistry_section_2

Depending on the tissue type and the method of antigen detection, endogenous biotin or enzymes may need to be blocked or quenched, respectively, prior to antibody staining. Immunohistochemistry_sentence_19

Although antibodies show preferential avidity for specific epitopes, they may partially or weakly bind to sites on nonspecific proteins (also called reactive sites) that are similar to the cognate binding sites on the target antigen. Immunohistochemistry_sentence_20

A great amount of non-specific binding causes high background staining which will mask the detection of the target antigen. Immunohistochemistry_sentence_21

To reduce background staining in IHC, ICC and other immunostaining methods, samples are incubated with a buffer that blocks the reactive sites to which the primary or secondary antibodies may otherwise bind. Immunohistochemistry_sentence_22

Common blocking buffers include normal serum, non-fat dry milk, BSA, or gelatin. Immunohistochemistry_sentence_23

Commercial blocking buffers with proprietary formulations are available for greater efficiency. Immunohistochemistry_sentence_24

Methods to eliminate background staining include dilution of the primary or secondary antibodies, changing the time or temperature of incubation, and using a different detection system or different primary antibody. Immunohistochemistry_sentence_25

Quality control should as a minimum include a tissue known to express the antigen as a positive control and negative controls of tissue known not to express the antigen, as well as the test tissue probed in the same way with omission of the primary antibody (or better, absorption of the primary antibody). Immunohistochemistry_sentence_26

Sample labeling Immunohistochemistry_section_3

Antibody types Immunohistochemistry_section_4

The antibodies used for specific detection can be polyclonal or monoclonal. Immunohistochemistry_sentence_27

Polyclonal antibodies are made by injecting animals with the protein of interest, or a peptide fragment and, after a secondary immune response is stimulated, isolating antibodies from whole serum. Immunohistochemistry_sentence_28

Thus, polyclonal antibodies are a heterogeneous mix of antibodies that recognize several epitopes. Immunohistochemistry_sentence_29

Monoclonal antibodies are made by injecting the animal and then taking a specific sample of immune tissue, isolating a parent cell, and using the resulting immortalized line to create antibodies. Immunohistochemistry_sentence_30

This causes the antibodies to show specificity for a single epitope. Immunohistochemistry_sentence_31

For immunohistochemical detection strategies, antibodies are classified as primary or secondary reagents. Immunohistochemistry_sentence_32

Primary antibodies are raised against an antigen of interest and are typically unconjugated (unlabeled), while secondary antibodies are raised against immunoglobulins of the primary antibody species. Immunohistochemistry_sentence_33

The secondary antibody is usually conjugated to a linker molecule, such as biotin, that then recruits reporter molecules, or the secondary antibody itself is directly bound to the reporter molecule. Immunohistochemistry_sentence_34

IHC reporters Immunohistochemistry_section_5

Reporter molecules vary based on the nature of the detection method, the most popular being chromogenic and fluorescence detection mediated by an enzyme or a fluorophore, respectively. Immunohistochemistry_sentence_35

With chromogenic reporters, an enzyme label reacts with a substrate to yield an intensely colored product that can be analyzed with an ordinary light microscope. Immunohistochemistry_sentence_36

While the list of enzyme substrates is extensive, alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes used most extensively as labels for protein detection. Immunohistochemistry_sentence_37

An array of chromogenic, fluorogenic and chemiluminescent substrates is available for use with either enzyme, including DAB or BCIP/NBT, which produce a brown or purple staining, respectively, wherever the enzymes are bound. Immunohistochemistry_sentence_38

Reaction with DAB can be enhanced using nickel, producing a deep purple/black staining. Immunohistochemistry_sentence_39

Fluorescent reporters are small, organic molecules used for IHC detection and traditionally include FITC, TRITC and AMCA, while commercial derivatives, including the Alexa Fluors and Dylight Fluors, show similar enhanced performance but vary in price. Immunohistochemistry_sentence_40

For chromogenic and fluorescent detection methods, densitometric analysis of the signal can provide semi- and fully quantitative data, respectively, to correlate the level of reporter signal to the level of protein expression or localization. Immunohistochemistry_sentence_41

Target antigen detection methods Immunohistochemistry_section_6

The direct method is a one-step staining method and involves a labeled antibody (e.g. FITC-conjugated antiserum) reacting directly with the antigen in tissue sections. Immunohistochemistry_sentence_42

While this technique utilizes only one antibody and therefore is simple and rapid, the sensitivity is lower due to little signal amplification, in contrast to indirect approaches. Immunohistochemistry_sentence_43

However, this strategy is used less frequently than its multi-phase counterpart. Immunohistochemistry_sentence_44

The indirect method involves an unlabeled primary antibody (first layer) that binds to the target antigen in the tissue and a labeled secondary antibody (second layer) that reacts with the primary antibody. Immunohistochemistry_sentence_45

As mentioned above, the secondary antibody must be raised against the IgG of the animal species in which the primary antibody has been raised. Immunohistochemistry_sentence_46

This method is more sensitive than direct detection strategies because of signal amplification due to the binding of several secondary antibodies to each primary antibody if the secondary antibody is conjugated to the fluorescent or enzyme reporter. Immunohistochemistry_sentence_47

Further amplification can be achieved if the secondary antibody is conjugated to several biotin molecules, which can recruit complexes of avidin-, streptavidin- or NeutrAvidin protein-bound enzyme. Immunohistochemistry_sentence_48

The difference between these three biotin-binding proteins is their individual binding affinity to endogenous tissue targets leading to nonspecific binding and high background; the ranking of these proteins based on their nonspecific binding affinities, from highest to lowest, is: 1) avidin, 2) streptavidin and 3) NeutrAvidin protein. Immunohistochemistry_sentence_49

The indirect method, aside from its greater sensitivity, also has the advantage that only a relatively small number of standard conjugated (labeled) secondary antibodies needs to be generated. Immunohistochemistry_sentence_50

For example, a labeled secondary antibody raised against rabbit IgG, which can be purchased "off the shelf", is useful with any primary antibody raised in rabbit. Immunohistochemistry_sentence_51

This is possible because all rabbit IgG in this example would have the same Fc (constant) region, thus even with a small number of anti-rabbit secondary antibodies generated, each could adhere to any primary rabbit antibody. Immunohistochemistry_sentence_52

This is particularly useful when a researcher is labeling more than one primary antibody, whether due to polyclonal selection producing an array of primary antibodies for a singular antigen or when there is interest in multiple antigens. Immunohistochemistry_sentence_53

With the direct method, it would be necessary to label each primary antibody for every antigen of interest. Immunohistochemistry_sentence_54

Counterstains Immunohistochemistry_section_7

After immunohistochemical staining of the target antigen, a second stain is often applied to provide contrast that helps the primary stain stand out. Immunohistochemistry_sentence_55

Many of these stains show specificity for specific classes of biomolecules, while others will stain the whole cell. Immunohistochemistry_sentence_56

Both chromogenic and fluorescent dyes are available for IHC to provide a vast array of reagents to fit every experimental design, and include: hematoxylin, Hoechst stain and DAPI are commonly used. Immunohistochemistry_sentence_57

Troubleshooting Immunohistochemistry_section_8

In immunohistochemical techniques, there are several steps prior to the final staining of the tissue antigen, which can cause a variety of problems including strong background staining, weak target antigen staining, and autofluorescence. Immunohistochemistry_sentence_58

Endogenous biotin or reporter enzymes or primary/secondary antibody cross-reactivity are common causes of strong background staining, while weak staining may be caused by poor enzyme activity or primary antibody potency. Immunohistochemistry_sentence_59

Furthermore, autofluorescence may be due to the nature of the tissue or the fixation method. Immunohistochemistry_sentence_60

These aspects of IHC tissue prep and antibody staining must be systematically addressed to identify and overcome staining issues. Immunohistochemistry_sentence_61

Diagnostic IHC markers Immunohistochemistry_section_9

IHC is an excellent detection technique and has the tremendous advantage of being able to show exactly where a given protein is located within the tissue examined. Immunohistochemistry_sentence_62

It is also an effective way to examine the tissues. Immunohistochemistry_sentence_63

This has made it a widely used technique in the neurosciences, enabling researchers to examine protein expression within specific brain structures. Immunohistochemistry_sentence_64

Its major disadvantage is that, unlike immunoblotting techniques where staining is checked against a molecular weight ladder, it is impossible to show in IHC that the staining corresponds with the protein of interest. Immunohistochemistry_sentence_65

For this reason, primary antibodies must be well-validated in a Western Blot or similar procedure. Immunohistochemistry_sentence_66

The technique is even more widely used in diagnostic surgical pathology for immunophenotyping tumors (e.g. immunostaining for e-cadherin to differentiate between DCIS (ductal carcinoma in situ: stains positive) and LCIS (lobular carcinoma in situ: does not stain positive)). Immunohistochemistry_sentence_67

More recently, Immunohistochemical techniques have been useful in differential diagnoses of multiple forms of salivary gland, head, and neck carcinomas. Immunohistochemistry_sentence_68

The diversity of IHC markers used in diagnostic surgical pathology is substantial. Immunohistochemistry_sentence_69

Many clinical laboratories in tertiary hospitals will have menus of over 200 antibodies used as diagnostic, prognostic and predictive biomarkers. Immunohistochemistry_sentence_70

Examples of some commonly used markers include: Immunohistochemistry_sentence_71



Directing therapy Immunohistochemistry_section_10

A variety of molecular pathways are altered in cancer and some of the alterations can be targeted in cancer therapy. Immunohistochemistry_sentence_72

Immunohistochemistry can be used to assess which tumors are likely to respond to therapy, by detecting the presence or elevated levels of the molecular target. Immunohistochemistry_sentence_73

Chemical inhibitors Immunohistochemistry_section_11

Tumor biology allows for a number of potential intracellular targets. Immunohistochemistry_sentence_74

Many tumors are hormone dependent. Immunohistochemistry_sentence_75

The presence of hormone receptors can be used to determine if a tumor is potentially responsive to antihormonal therapy. Immunohistochemistry_sentence_76

One of the first therapies was the antiestrogen, tamoxifen, used to treat breast cancer. Immunohistochemistry_sentence_77

Such hormone receptors can be detected by immunohistochemistry. Immunohistochemistry_sentence_78

Imatinib, an intracellular tyrosine kinase inhibitor, was developed to treat chronic myelogenous leukemia, a disease characterized by the formation of a specific abnormal tyrosine kinase. Immunohistochemistry_sentence_79

Imitanib has proven effective in tumors that express other tyrosine kinases, most notably KIT. Immunohistochemistry_sentence_80

Most gastrointestinal stromal tumors express KIT, which can be detected by immunohistochemistry. Immunohistochemistry_sentence_81

Monoclonal antibodies Immunohistochemistry_section_12

Main article: Monoclonal antibody therapy Immunohistochemistry_sentence_82

Many proteins shown to be highly upregulated in pathological states by immunohistochemistry are potential targets for therapies utilising monoclonal antibodies. Immunohistochemistry_sentence_83

Monoclonal antibodies, due to their size, are utilized against cell surface targets. Immunohistochemistry_sentence_84

Among the overexpressed targets are members of the EGFR family, transmembrane proteins with an extracellular receptor domain regulating an intracellular tyrosine kinase. Immunohistochemistry_sentence_85

Of these, HER2/neu (also known as Erb-B2) was the first to be developed. Immunohistochemistry_sentence_86

The molecule is highly expressed in a variety of cancer cell types, most notably breast cancer. Immunohistochemistry_sentence_87

As such, antibodies against HER2/neu have been FDA approved for clinical treatment of cancer under the drug name Herceptin. Immunohistochemistry_sentence_88

There are commercially available immunohistochemical tests, Dako HercepTest, Leica Biosystems Oracle and Ventana Pathway. Immunohistochemistry_sentence_89

Similarly, EGFR (HER-1) is overexpressed in a variety of cancers including head and neck and colon. Immunohistochemistry_sentence_90

Immunohistochemistry is used to determine patients who may benefit from therapeutic antibodies such as Erbitux (cetuximab). Immunohistochemistry_sentence_91

Commercial systems to detect EGFR by immunohistochemistry include the Dako pharmDx. Immunohistochemistry_sentence_92

Mapping protein expression Immunohistochemistry_section_13

Immunohistochemistry can also be used for a more general protein profiling, provided the availability of antibodies validated for immunohistochemistry. Immunohistochemistry_sentence_93

The Human Protein Atlas displays a map of protein expression in normal human organs and tissues. Immunohistochemistry_sentence_94

The combination of immunohistochemistry and tissue microarrays provides protein expression patterns in a large number of different tissue types. Immunohistochemistry_sentence_95

Immunohistochemistry is also used for protein profiling in the most common forms of human cancer. Immunohistochemistry_sentence_96

See also Immunohistochemistry_section_14


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