| IHC DETECTION SYSTEMS |
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What is IHC?
IHC stands for Immunohistochemistry. Immunohistochemistry is a technique for localizing and visualizing an antigen in a tissue section by using an antibody specific for the target antigen. The immunohistochemistry procedure consists of tissue preparation, antibody incubation, and a series of detection reactions. Technically, when the procedure is performed on cells (cell smears, cytospins, etc.) rather than tissue sections, it is termed immunocytochemistry, but in practice the two terms are often used interchangeably.
What is the principle of the IHC Detection System Kit?
A two-stage process detects antigens in tissues and cells - The binding of the primary antibody to a specific epitope and the subsequent detection of this binding by a colorimetric reaction.
Tissues or cell preparations are frozen or fixed, sectioned, and attached to slides. The sections are then dewaxed if paraffin-embedded, treated with a target retrieval solution if required, blocked with a protein-based blocking solution and then incubated with a primary antibody. The bound primary antibody is detected by adding biotinylated secondary antibody, enzyme-streptavidin conjugate, and substrate. When adequate color development is seen, the slides are washed in water to stop the reaction, counterstained, and covered with a mounting medium.
How can one localize antigens on tissues?
There are two methods to localize antigens on tissues - via labeled (i.e. avidin-biotin) and unlabeled (i.e. enzyme-anti-enzyme). The labeled method can be used directly or indirectly, while the unlabeled method is always used indirectly.
Direct Method: In this method, an enzyme is directly conjugated to the primary antibody before it is applied to the tissue. The reaction is subsequently visualized by combining the substrate with a chromogen and applying it to the tissue. This method is used rather infrequently as the staining is often weak, and a separate conjugation reaction must be performed for each primary antibody used.
Indirect Method: Indirect detection systems offer improved sensitivity and reduced background over the direct method. The first step in this procedure is to apply the primary antibody to the tissue. A secondary antibody raised in another animal against the immunoglobulin type of the primary antibody is subsequently applied and binds to the primary. This secondary antibody is conjugated to an enzyme or fluorescent label. The reaction is visualized by applying a mixture of substrate and chromogen, which reacts with the conjugated enzyme to produce a color change.
The indirect method was further improved by separating the labeling enzyme from the link antibody. This method provided an additional level of signal amplification. The following systems are all variations in the three-step amplification method.
- Peroxidase-Anti-Peroxidase (PAP)
- Alkaline Phosphatase - Anti-alkaline Phosphatase (APAAP)
- Avidin-Biotin Complex (ABC)
- Biotin-Streptavidin Amplified (B-SA)
- Enhanced Biotin-Streptavidin (B-SA)
- Non-Biotin HRP Polymer
How does the Streptavidin-Biotin (also known as Link-Label) Detection System work?
The BioGenex Link-Label Detection System uses the streptavidin-biotin technology. The method relies on the high binding affinity of Streptavidin for Biotin. Streptavidin is a tetrameric 60 kD Avidin analog isolated from the bacterium Streptomyces avidinii capable of binding four molecules of biotin with a very high affinity.
This affinity is approximately 106 times greater than that of most antibodies for their antigens, and provides very specific detection and amplification of the antigen-antibody-binding event. In the Biotin-Streptavidin system, after blocking endogenous enzyme and non-specific protein binding sites in the tissue, the sections are incubated with a primary antibody specific for the antigen to be demonstrated.
The sections are then incubated with linking antibody (a biotinylated immunoglobulin such as anti-mouse lg). Next, the labeling reagent (an enzyme-labeled streptavidin) binds to the biotin residues on the link antibody.
The entire antibody-enzyme complex is then made visible by the addition of a chromogenic substrate.
What are the steps of the protocol for the Link-Label IHC Detection system?
- Remove wax with EZ-DeWax™ Solution
- Retrieve antigen with Antigen Retrieval Solutions(need-basis)
- Inactivate endogenous peroxidase by incubation with Peroxide Block (for HRP)
- Minimize non-specific binding by incubation with Power Block™ Reagent
- Apply Primary Antibody and incubate
- Apply biotinylated Secondary Antibody and incubate (Link)
- Apply Streptavidin-Enzyme conjugate and incubate (Label)
- Apply Substrate Solution and incubate until color is developed
- Counterstain the tissue with Hematoxylin or Nuclear Fast Red
- Mount the tissue with Mounting Medium
- Examine the slide by appropriate microscopy
How does the Polymer-HRP IHC Detection System work?
The Polymer-HRP IHC Detection System uses a non-biotin polymeric technology. This system is highly recommended in those cases where endogenous biotin represents a major source of background. Since the system is not based on the conventional streptavidin-biotin chemistry, this problem is completely eliminated. This detection system uses an advanced non-biotin polymeric technology. It consists of two major components:
Super Block™ - A universal blocking reagent for saturating positive charges in tissue sections; and
Poly-HRP Reagent - A multivalent secondary antibody HRP-polymer conjugate, that detects rabbit and mouse primary IgG. As a result, this non-biotin system achieves better signal amplification, thereby enhancing the sensitivity and specificity of IHC detection.
What are the steps of the protocol for the Polymer-HRP IHC Detection system?
- Remove wax with EZ-DeWax™ Solution
- Retrieve antigen with Antigen Retrieval Solutions(need-basis)
- Inactivate endogenous peroxidase by incubation with Peroxide Block (for HRP)
- Minimize non-specific binding by incubation with Power Block™ Reagent
- Apply Primary Antibody and incubate
- Apply SuperBlock™ and incubate
- Add Poly-HRP Reagent and incubate
- Apply Substrate Solution and incubate until color is developed
- Counterstain the tissue with Hematoxylin
- Mount the tissue with Mounting Medium
- Examine the slide by appropriate microscopy
Which types of chromogenic substrates may be used with HRP tests?
The type of chromogenic substrate depends upon the type of enzyme used, e.g. AEC and DAB can be used with horseradish peroxidase. Fast Red and New Fuchsin can used with alkaline phosphatase.
Substrates with HRP Tests:
AEC (3-amino-9-ethylcarbazole) forms a red end product. This substrate is alcohol soluble, hence aqueous hematoxylin and an aqueous mounting medium must be used. Staining is semi-permanent, but is susceptible to oxidation. Although it may fade after an extended period of time; clear nail polish can be applied around the edges of the cover slip as a seal, rendering it permanent.
DAB (3,3-diaminobendizidine tetrahydrochloride) yields a brown colored end product, which is permanent and resistant to alcohol and xylene. DAB must be used with great care and proper precautions, as it is a potential carcinogen.
Substrates with Alkaline Phosphatase Tests:
Fast Red forms a red fluorescent end product. As with AEC, this substrate is also alcohol soluble. The same care should be taken to avoid dissolving the color precipitate of this chromogen.
New Fuschsin is a pinkish red chromogen. It may be considered to be a permanent stain but has been found to fade in alcohol. It may be permanently mounted by placing it only in xylene after air-drying. A cover slip with a permanent mounting media may then be used. It may also be mounted with SuperMount in which case no cover slip is required.
What are the advantages of the IHC Detection System?
BioGenex IHC systems have the following advantages:
- Matched detection reagents and protocols for maximal signal-to-noise ratios
- Very high specificity and sensitivity
- Publication-quality results in minimum time.
How does sample type affect staining?
Type of sample and how it affects staining:
Monolayers: Either cells grown on a solid substrate, cytospins or smears can give
weak staining if proper permeabilization is not performed. Lipids in cell membrane will interfere in the hybridization of the probe. Fixation with acetone and/or methanol will remove those lipids. The proteolytic treatment with Protease K also helps in the permeabilization of the membranes.
Tissue Sections: Blocks of tissues embedded in wax or frozen and the cut into thin
sections, or tissue imprints. Weak staining in this case can be the result of only a part of the
cell being available for the detection of the target. One solution is cutting thicker sections, but not too thick. Adequate permeabilization is achieved by pretreatment with protease or Antigen Retrieval
What IHC controls should one set up for conducting the experiment?
A set of controls should be run with the antibody and tissue to be tested. These controls are for initial testing only. More controls may be needed for troubleshooting purposes.
Negative controls: without primary antibody, without secondary antibody, and without streptavidin-enzyme conjugates.
Positive controls: a positive antibody with the test tissue, or the test antibody with a positive tissue.
TROUBLESHOOTING TIPS
Why does my negative control show strong signal?
The signal is due to non-specific cross-reactivity of detection reagents.
| Negative Control | Possible Cause of Signal | Solution |
| Only Primary antibody is omitted. |
The secondary antibody is binding non-specifically to the tissue. |
Add 0.1% tissue-specific serum to the secondary antibody.
Dilute the secondary antibody.
Change species of secondary antibody.
Change the incubation time for both substrate and secondary antibody
Ensure no drying has taken place |
| Only secondary antibody is omitted. |
The streptavidin-enzyme conjugate is binding non-specifically to the tissue. |
Block tissue with the Avidin/Biotin Blocking Reagents. |
| Only streptavidin-enzyme conjugate is omitted. |
Intrinsic tissue enzyme activity is interfering with the reaction |
Treat tissue with hydrogen peroxide solution (for HRP) or add levamisole to substrate (for Alk Phos). |
Why does my positive control have no signal?
In most cases, this is because the antibody is not optimized or the tissue is not adequately treated.
| Positive Controls |
Possible Cause for No Signal |
Solution |
| Using an antibody known to react with the test tissue, or using the test antibody with cells known to contain the antigen. |
Fixatives may have reduced access of antibody to antigen. |
Perform microwave target retrieval procedure or protease digestion. |
| Antibody may be too dilute. |
Titrate antibody to determine the optimum dilution that gives the best signal-to-noise ratio. |
| Secondary antibody does not recognize the primary antibody. |
Ensure biotinylated secondary antibody is directed against the species of primary antibody, e.g. anti-mouse secondary antibody for mouse primary antibody. |
| The enzyme/substrate system is defective or incompatible. |
This can be confirmed by performing the Dot Blot Test (see instruction manual). |
What references could you provide if one wants to learn more about IHC?
Carson, F.L. (1990) Histotechnology: A Self-Instructional Text. ASCP Press, Chicago.
Elias, J.M. (1990) Immunohistopathology: A Practical Approach to Diagnosis.
ASCP Press, Chicago.
Taylor, C.R. and Cote, R.J. (1994) Immunomicroscopy: A Diagnostic Tool for the Surgical Pathologist. W.B. Saunders Co., Philadelphia.
Shi, S.R., Gu, J., Kalra, K.L., Chen, T., Cote, R.J., and Taylor, C.R. (1995) Antigen retrieval technique: a novel approach to immunohistochemistry on routinely processed tissue sections. Cell Vision 2: 6-22.
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