Group 3 Project- Immunohistochemistry

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What is Immunohistochemistry?

Immunohistochemistry is a technique that is regularly used in cell biology, biological research and diagnostic pathology. It relies upon the interaction between antibodies and antigens, allowing for substances to be identified within tissue samples. An antigen is a substance that is recognised by the immune system as foreign, prompting the production of antibodies and causing an immune response within the body. Antigens are usually large, complex proteins or polysaccharide molecules (M. A. Hayat, 2002). An antibody is a glycoprotein that is produced by B lymphocytes in response to the presence of an antigen. Also called immunglobulins, antibodies neutralise or destroy antigens by binding to them in a specific interaction, made possible by a 'lock and key' mechanism, where specific antibodies are only able to bind with specific antigens.

Immunohistochemistry (IHC) is a technique used to locate antigens or proteins in tissue sections. It utilises this antigen-antibody interaction by labelling antibodies which will react with specific antigens. This interaction is visualised by a marker which may be a fluorescent dye, enzymes, radioactive elements or even colloidal gold.

The Process

Immunohistochemistry has a general process which differs slightly depending on which method is utilised. These are the steps involved:

1. A tissue sample is collected from an animal or the patient. It can be from almost any organ in the body.

2. The sample must be frozen or preserved quickly to prevent deterioration of the tissues. Fresh samples must be used as soon as possible. This is known as the fixation process.

3. Frozen samples are sliced to one-cell thickness and mounted.

4. Antibodies are added to the sample, which bind with the antigens present in the tissue. A protein solution is added to prevent the antibodies binding to non-specific proteins in a process called blocking.

5. The sample is then incubated and washed to remove excess primary antibodies.

6. A secondary antibody is added to the sample and similarly to previous steps, it is incubated and washed to remove any excess secondary antibodies.

7. After mounting, these antibodies are fluorescently tagged and are visualized with a microscope.

There are various different methods including direct, indirect, PAP, ABC and LSAB.


1941 - Albert H. Coons first introduced immunofluorescence as initial attempts to label antibodies were unsuccessful as the labels were not visible enough under the microscope. Using specific antibodies, Coons labeled them with fluorescent dyes in order to localise substances in tissues. This allowed for the detection of antibodies, antigens and antigenic proteins in tissues. <ref> Mao, Su-Yau, Javois, Lorette C., Kent, Ute M. "Overview of Antibody Use in Immunocytochemistry" From: Methods in Molecular Bology, Vol. 115: Immunocytochemical Methods and Protocols, Edited by L. C. Javois, Humana Press Inc, Totowa, New Jersey Cite error: The opening <ref> tag is malformed or has a bad name

1942 - Albert H. Coons, Hugh Creech, Norman Jones and Ernst Berliner succeeded in tagging antibodies and using these antibodies to detect foreign antigens in tissues. This involved using a single antipneumococcal antibody to find pnuemococcal antigens in mice injected with large numbers of pneumococci. (


Direct Method

Direct fluorescent methods is one of the oldest and simplest methods. It utilises one fluorescently-labelled antibody which reacts directly with an antigen within a tissue sample. This method is rarely used since the introduction of more complicated modern techniques.




Indirect Method




PAP Method




ABC Method

The ABC method is an indirect method of immunohistochemistry. It stands for the Avidin-Biotin Complex Method.

Firstly, the tissue of interest is sectioned and is incubated with a primary antiserum that targets the antigen we would like to locate in the tissue section. An antiserum is a serum containing antibodies such as agglutinins and antitoxins. This antiserum is known as the primary antibody and is injected into the tissue to target the antigen and causes an antibody-antigen reaction. After the primary antibody is inserted. A secondary labeled antibody is added, namely a biotinylated antibody. The secondary antibody reacts with the first antibody and launches a large mass of biotin into the area in which the antigen is situated. The secondary antibody is reacts in an opposite manner to the primary antibody. The secondary antibody does not have the intention in reacting with the antigen.

The addition of the avidin biotin enzyme complex causes binding to the secondary antibody. The avidin biotin enzyme complex consists of avidin, biotin and enzymes. Avidin has a high attraction for biotin with four uniting sites in each molecule. Enzymes readily bind to biotin and it is because of those two properties that enables the formation of the avidin biotin complex. The last step in the formation of the complex before it is ready to use is by combining it with a solution. The avidin biotin complex is inserted into the tissue and the biotinylated secondary body that is already attached to the antigen binds to any free biotin sites on the avidin molecule.

The last step of the procedure is the addition of an enzyme substrate to the tissue section. This acts as a marker for site of the antibody-antigen reaction. The ABC method increases the influx of enzymes which increases the efficiency in detecting the antigen.


Since there is an large influx of enzymes into the tissue section, it increases the efficiency in detecting the antigen. This method does not require a large amount of primary antibody. A light concentration of primary antibody has shown to produce a stronger stain than methods using the same concentration of antibody. The process of ABC method is very fast, it usually can be completed within less than three hours. Once the avidin biotin complex is assembled it can be used for several days because it maintains a stable condition for that period of time.


( more coming )

Blocking of biotin is needed.


LSAB Method

Comparison Between Methods

Current Research/Breakthroughs



2010 Projects

Fluorescent-PCR | RNA Interference | Immunohistochemistry | Cell Culture | Electron Microsopy | Confocal Microscopy | Monoclonal Antibodies | Microarray | Fluorescent Proteins | Somatic Cell Nuclear Transfer