Group 3 Project- Immunohistochemistry

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What is Immunohistochemistry?

Example of 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. 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. [1]

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.

This method varies somewhat as there are different immunohistochemistry methods including; Direct and Indirect Method, Peroxidase-Antiperoxidase (PAP) Method, Avidin-antibiotin complex (ABC) Method, Labeled StreptAvidin Biotin (LSAB) Method, Polymeric Method and Catalysed Signal Amplification (CSA) Methods.


Like most experiments, a control needs to be run so that the procedure can be verified and to check that the antibody being used is the correct one. The controls should be handled and processed in the same way the tissues being tested are, to ensure consistency and accurate results. A control should be set up for every set of experimental conditions.

There are two types of control in Immunohistochemistry; Positive control and Negative control.

1. Positive control is used to verify the procedure. Any negative results or steps that do not work efficiently will be eliminated or completed over again until the correct procedure and staining is established. Generally it is most appropriate to use the tissue which will be used in the experiment, as the most accurate results will assist in modifying the procedure making the experimental results the most reliable.

2. Negative staining is used to verify the specificity of the antibody being used. The antibody must be from the same species as the primary antibody and a stain must not occur when the primary antibody is removed or replaced with serum. This type of negative control is often used in IHC as it is easy.

A second type of negative staining is used, in which the staining is inhibited by a purified antigen absorbing the primary antibody. This technique is the most useful and appropriate, however isolating a purified antigen is time consuming and hard, consequently making this stain rarely used in Immunohistochemistry controls.[2]


The process called blocking, is used to reduce or limit the amount of background staining which occurs during various Immunohistochemistry methods. Background staining is when cells that are not being stained for pick up some of the stain or fluorescence, leading to false positives. This is a common outcome when fixation is not completed quickly or adequately enough and also in the middle of large pieces of tissue. Background staining may also occur if the antibodies being used have been contaminated by other antibodies, through the use of impure antigen during immunization.

Background staining has two types, specific or non-specific.

Non-specific staining is when the antibodies bind to non-specific proteins instead of the antigens they are supposed to. The uniform stain that occurs can be limited by blocking the sites with normal serum.

Specific staining is when activity of a similar antigen within a tissue is similar to what is being added to the tissue overrides the experiment as all of the antibodies are taken up but not by the sites wanted. An example of this is during the use of the Avidin-antibiotin complex (ABC) Method. Some liver and kidney tissues have natural biotin. To stop the avidin binding with this biotin, the tissue is pretreated with unconjugated avidin and then biotin is added until saturation.

Natural fluorescence that exists within some tissues can interfere with the used of added fluorescent dyes, resulting in background staining. Generally it is best to use enzyme labeling methods with such tissues.[3]


Albert Coons - Courtesy of the Harvard Medical School Countway Library

1941 - Albert H. Coons

Coons first introduces 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. [4]

1942 - Albert H. Coons, Hugh Creech, Norman Jones and Ernst Berliner

Coons, Creech, Jones and Berliner succeeded in tagging antibodies. These antibodies were used 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. [5]

1959 - Singer

Singer first used an electron-dense protein in order to achieve ultrastructural localisation. The protein ferritin was used to tag an antibody. Electron microscopy could be used in immunohistochemistry as a result of this as the presence of iron in the protein makes it electron-dense. [6]

1965 - Sternberger

Uranium was used to develop the first electron-opaque heavy metal technique. [7]

1966 - Graham and Karnovsky

First localised the enzyme peroxidase using cytochemical methods leading to the development of the enzyme tagging method. [8]

1967 - Nakane and Pierce

Nakane and Pierce developed the enzyme-labelled antibody technique by labelling an antibody with an enzyme.[9]

1970 - Sternberger

Building upon the work of Graham, Karnovsky, Nakane and Pierce, Sternberger developed the peroxidase-antiperoxidase (PAP) method in an attempt to improve the enzyme-labelled method. The PAP method was an unlabelled antibody method. [10]

1971 - Faulk and Taylor

Another electron-opaque heavy metal technique was developed by Faulk and Taylor using colloidal gold. This is a popular technique and can also be called the colloidal gold technique. [11]

1974 - Heitzman and Richards

The Avidin-antibiotin complex (ABC) method was developed. Similar to the PAP method, it is also an unlabelled antibody method. [12]

1990's - Antigen Retrieval

It was discovered that the retrieval of nonreactive antigens in formalin-fixed, paraffin-embedded tissues was possible by heating sections in buffer solutions. This increased the detection of antigens and sensitivity of methods.[13]


Direct Method

Simplified drawing of Direct Method

Direct fluorescent methods is the oldest and simplest method. It utilises one labelled primary antibody which reacts directly with an antigen within a tissue sample. A sample is prepared and is exposed to a primary antibody. The antibodies react with the antigens resulting in an antigen-antibody interaction. The sample is washed to remove excess antibodies and is mounted and visualised under a microscope. This method is rarely used since the introduction of more complicated modern techniques.[14]

Advantages Disadvantages
Procedure is short and quick. Procedure is insensitive.
It can be used for quick diagnostic testing. Only one tagged antibody binds with each antigen. If antigen concentration is low then the the concentration of tagged antibodies is low and may not be enough for detection under the microscope.

Since the introduction of the more accurate and sensitive indirect method, the direct method is rarely used.

Indirect Method

Simplified drawing of Indirect Method

The indirect method is used far more commonly than the direct method. It involves using both primary and secondary antibodies. Similarly to the direct method, the sample is exposed to primary antibodies and the antigens in the sample react with the antibodies resulting in an antigen-antibody interaction. The sample is washed to remove the excess antibodies and is then exposed to the labelled secondary antibodies which are directed against the primary antibodies causing them to bind together. The sample is mounted and can be visualised through a microscope.

The secondary antibodies may be labelled with various substances. If they are labelled with fluorescent dyes such as Texas Red, rhodamine or FITC the method becomes known as Indirect Immunofluorescence Method. Alternatively, they may be labelled with enzymes such as peroxidase or glucose oxidase. This is known as Indirect Immunoenzyme Method. [15]

Advantages Disadvantages
Only the secondary antibodies, which tend to be cheaper, need to be labelled, thus preventing wastage of primary antibodies. Secondary antibodies must be from a different animal species
Sensitivity is much greater than that of direct method Procedure is laborious

The indirect method acts as a precursor to more complex methods such as the PAP method and ABC method. It is not as commonly used as its more complex successors.

Peroxidase-Antiperoxidase (PAP) Method

Simplified drawing of PAP Method

The PAP method was pioneered by Sternberger in the 1970's and is a development of the indirect technique. Similar to the indirect method, the PAP method exposes an unlabelled primary antibody to the antigens in the sample. Following this, the sample is rinsed to remove any excess primary antibodies, allowing for a secondary antibody to be introduced to the sample. The primary antibodies react with the antigens in an antigen-antibody reaction, binding them together. The secondary antibodies react with the primary antibodies. [16]

Following this step and unique to the PAP method is the introduction of the PAP molecule. Horseradish peroxidase acts as an antigen when injected into an animal and when combined with immunoglobins, creates a stable antigen-antibody complex, known as the PAP complex. As this combination does not damage enzyme activity, the PAP complex is a versatile tool for the detection of binding sites of anti-antibodies. [17]

The PAP complex acts as an antigen and reacts with the secondary antibodies, making up the third layer. The sample is then visualised under a microscope. This multiple layer method is most commonly used in diagnostic laboratories working with formalin-fixed, paraffin-embedded section. [18]

Advantages Disadvantages
Sensitivity is between 100-1000 times greater than the indirect method [19] The primary antibody and the PAP complex must be from the same species [20]
Allows for the primary antibody to be more diluted
Reduces non-specific background staining

PAP method is used in both diagnostics and research due to its high specificity and sensitivity. Recent uses include:

  • Proliferation, steroid receptors and clinical/pathological response in breast cancer treated with letrozole [21]
  • c-kit Expression in small cell carcinoma of the urinary bladder: prognostic and therapeutic implications [22]

Avidin-Biotin Complex Method (ABC Method)

The ABC method is an indirect method of immunohistochemistry.

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. [23] [24]

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. [25] [26]

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.

Advantages Disadvantages
Increases efficiency in detecting the antigen when a secondary antibody is used Blocking of biotin is needed to prevent the non specific binding of avidin to endogenous biotin
Does not require a large amount of primary antibody On several occasion, the complex produced can be too big to seep into the tissue
Process is very fast (within three hours) Endogenous biotin can cause non specific staining.
Once the avidin biotin complex is assembled it can be used for several days

Image002.gif Image003.jpg

Example of an application of the ABC method

In 2003, there was an epidemic outbreak of highly pathogenic avian influenze ( HPAI) in Southeast Asia. This outbreak was caused by the viruses of the H5N1 virus. Humans were slowly being infected by this virus and the main reason for their infection was due to direct contact with birds that were infected with the H5N1 HPAI virus. In order to understand where this particular viral influenza antigen lesions lie within the birds, the ABC method was applied to detect the antigen within the birds. The tissue of interest was sectioned off and stained. A rabbit antinucleoprotein serum was inserted into the tissue section as the primary antibody. A biotinylated goat anti-rabbit IgG1 was applied as the secondary antibody. The ABC complex was then added. Lastly, an enzyme substrate called the substrate 3-amino-9-ethylcarbazole was introduced. The overall process produced a bright red indicator at the site of antigen. It was found that the main locations for these lesions are in the pancreas, brain and liver. [28]

Labeled StreptAvidin Biotin (LSAB) Method

This method is an improved method of the ABC method and because of this it overcomes some of the disadvantages that the ABC method poses. The lSAB method replaces the avidin used in the ABC method with streptavidin. Streptavidin is similar to avidin in structure and is tetrameric. Steptavidin has a high capability in binding biotin.

The method works similar to the ABC method. Primary antibody is added which binds to the antigen. Secondary antibody is added and the biotinylated antibody binds to the primary antibody. Streptavidin molecule binds to enzyme molecules directly and this complex is injected into the tissue section and penetrates through to the site of antigen. Peroxidase or alkaline phosphatase is added in the next step to enable the detection of the enzyme location.

There are a few reasons why this method has its advantage over the ABC method. The advantages stems from the fact that streptavidin has more advantages over avidin. Unlike avidin, streptavidin does not contains carbohydrates which prevents the non-specific binding of lectin-like molecules which are found naturlly in the body in the kidney,brain and liver. Avidin has an isoelectric point of 10 whereas strepavidin has an isoelectric point which is close to neutral. This neutral isoelectric point is beneficial because it does not elicit the non specific electrostatic binding of the streptavidin to natural tissue cells. The stablity of the complex used in the LSAB method is alot higher than the complex used in the ABC method due to the fact that streptavidin is conjugated directly to the enzyme. This allows the complex to be stored for a much longer period of time than the ABC complex. [29]

Polymeric Methods

Polymeric image.GIF

These methods were developed in search for immunohistochemistry methods that are both reliable and does not consume too many steps. It is a two step labelling method. An example of a polymer enhanced method is the EnVision method. In this technique, the primary antibody is applied to the tissue section, proceeded by the application of polymeric conjugates. The polymeric conjugates are made up centrally of a dextran backbone in which secondary antibodies and perioxidase binds onto. Each dextran backbone binds approximately 100 enzyme molecules and 20 antibody molecules.

When the polymeric conjugates is added, the secondary antibodies contained within it binds to the primary antibody which has been attached to the antigen. The enzyme attached to the polymer conjugate helps detect the location of the antigen within the tissue section by staining. [30] [31]

Advantages Disadvantages
It is a two step process which therefore is more time efficient The high molecular weight of the backbone can reduce the efficiency of penetration into the tissue


A visual representation of the Sensitivity of Immunohistochemistry methods from least sensitive to most sensitive

Immunohistochemistry has numerous methods that can be used in the lab, with some having substantial advantages over others. One such property, is sensitivity. The sensitivity of a technique is important as it dictates a method’s use and also the results that will be obtained. A range of sensitivities can be seen within the methods and generally the more sensitive the method, the better the results that will be obtained.

The spectrum of sensitivity in the methods can be seen clearly in the diagram on the right with the most sensitive at the top of the curve and the least at the bottom.

Direct and indirect methods were not included in this diagram as these techniques are the least sensitive and are rarely used, as more sensitive techniques are favoured.[32]


Antibody Glycoproteins that are produced by B lymphocytes in response to the presence of an antigen.

Antigen A complex protein or polysaccharide that is identified as a foreign substance within the body.

Immunocytochemistry Another name for immunohistochemistry.

Primary Antibody The antibody that reacts with the antigen in the sample; is generally unlabelled except when used in direct method.

Second Antibody The antibody which combines with the primary antibody; may or may not be labelled.


  1. Hayat M. A., Microscopy, Immunohistochemistry, and Antigen Retrieval Methods: For light and Electron Microscopy, Kluwer Academic/Plenum Publishers, New York, 2002, pp 31-33
  2. Introduction to Immunohistochemistry [1]
  3. Introduction to Immunohistochemistry [2]
  4. 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
  6. Bozzola,John J.,Russell,Lonnie Dee,Electron microscopy: principles and techniques for biologists,1999 [3]
  7. History of Immunohistochemistry [4]
  8. Bozzola,John J.,Russell,Lonnie Dee,Electron microscopy: principles and techniques for biologists, 1999 [5]
  9. Bozzola,John J.,Russell,Lonnie Dee,Electron microscopy: principles and techniques for biologists, 1999 [6]
  10. History of Immunohistochemistry [7]
  11. History of Immunohistochemistry [8]
  12. History of Immunohistochemistry [9]
  13. Ramos-Vara JA, Segalés J, Duran CO, et al. Diagnosing infectious porcine diseases using immunohistochemistry. Swine Health Prod. 1999;7(2):85–91.
  14. Introduction to Immunohistochemistry [10]
  15. Introduction to Immunohistochemistry [11]
  16. Introduction to Immunohistochemistry [12]
  17. Kiernan, J. A., Histological & Histochemical Methods: Theory and Practice, The Bath Press, Somerset, Third Edition, 1999
  18. Ramos-Vara JA, Segalés J, Duran CO, et al. Diagnosing infectious porcine diseases using immunohistochemistry. Swine Health and Production. 1999;7(2):85–91[13]
  19. Ramos-Vara JA, Segalés J, Duran CO, et al. Diagnosing infectious porcine diseases using immunohistochemistry. Swine Health and Production. 1999;7(2):85–91[14]
  20. Bratthauer, Gary L., The Peroxidase-Antiperoxidase (PAP) Method and other All-Immunologic Detection Methods, From: Methods in Molecular Bology, Vol. 115: Immunocytochemical Methods and Protocols, Edited by L. C. Javois, Humana Press Inc, Totowa, New Jersey
  21. W. R. Miller, S. White, J. M. Dixon, J. Murray, L. Renshaw, T. J. Anderson,Proliferation, steroid receptors and clinical/pathological response in breast cancer treated with letrozole, British Journal of Cancer (2006) 94, 1051–1056.doi:10.1038/sj.bjc.6603001 Published online 14 March 2006 [15]
  22. Chong-Xian Pan, Ximing J. Yang, Antonio Lopez-Beltran, Gregory T. MacLennan, John N. Eble, Michael O. Koch, Timothy D. Jones, Haiqun Lin, Kelly Nigro, Veronica Papavero, Maria Tretiakova,Liang Cheng,c-kit Expression in small cell carcinoma of the urinary bladder: prognostic and therapeutic implications, Modern Pathology (2005) 18, 320–323, advance online publication, 15 October 2004; doi:10.1038/modpathol.3800318. [16]
  23. Diagnostic Immunohistochemistry, David Dabbs second edition
  25. Diagnostic Immunohistochemistry, David Dabbs second edition
  28. Veterinery Pathology,Pathology of Natural Infections by H5N1 Highly Pathogenic Avian Influenza Virus in Mute (Cygnus olor) and Whooper (Cygnus cygnus) Swans J. P. Teifke1, R. Klopfleisch1, A. Globig1, E. Starick1, B. Hoffmann1, P. U. Wolf2, M. Beer1, T. C. Mettenleiter1 and T. C. Harder1 2007 doi: 10.1354/vp.44-2-137 vol. 44 no. 2 137-143
  29. Diagnostic Immunohistochemistry, David Dabbs second edition
  30. The EnVision++ system: a new immunohistochemical method for diagnostics and research. Critical comparison with the APAAP, ChemMate, CSA, LABC, and SABC techniques. Sabattini E, Bisgaard K, Ascani S, Poggi S, Piccioli M, Ceccarelli C, Pieri F, Fraternali-Orcioni G, Pileri SA. J Clin Pathol. 1998 Jul;51(7):506-11. PMID: 9797726
  31. Diagnostic Immunohistochemistry, David Dabbs second edition
  32. Introduction to Immunohistochemistry [17]

2010 Projects

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