Immunohistology Techniques

Immunohistochemical techniques utilize antibodies to detect and visualize cellular antigens in cells and tissue (in situ) using chromogenic or fluorescent methods. The immunohistochemist has a variety of standard tools to optimize the detection of a particular analyte. The direct method uses a labeled reagent, such as an antibody labeled with a fluorescent tag or an enzyme, which can be visualized conversion and deposition of substrate at the site of antigen-antibody interaction. Indirect methods increase the number of assay steps, but enable the user to amplifythe assay for improved sensitivity. For optimal sensitivity, a biotin-streptavidin or biotin-avidin method is recommended.

Direct Detection
Direct methods are appropriate when labeled primary antibodies are available or when the target molecule is present at levels that do not require amplification. In a direct assay, a fluorochrome-labeled or enzyme-labeled antibody reacts directly with the antigen in the tissue (Figure 1). The fluorochrome allows immediate visualization of the antigen with the use of a fluorescent microscope, while the enzyme requires the addition of a chromogenic substrate, followed by visualization using a light microscope. Use of a direct method is highly dependent upon the availability of the appropriately labeled primary antibody. Because this method utilizes only one antibody, it can be completed quickly and nonspecific reactions are minimized. However, this method is used less often because staining involves only one antibody and no amplification occurs.

Figure 1: Direct Detection Method:  Enzyme-labeled primary antibody reacts with tissue antigen.

Figure 2: Indirect Detection Method:  Enzyme-labeled secondary antibody binds to primary antibody bound to tissue antigen.

Indirect Detection
Indirect detection becomes necessary when the analyte is present in small amounts or when the required labeled primary antibody is not available. In a typical indirect assay, unconjugated primary antibody binds to the antigen. Then an enzyme-labeled secondary antibody binds to the primary antibody (Figure 2). Chromogenic substrate is added to provide visualization of the antigen. Signal amplification occurs because secondary antibodies bind to multiple epitopes on the primary antibody. Thus, more enzyme molecules are attached at the site of the antigen resulting in greater sensitivity. This method is more versatile than the direct method because a variety of primary antibodies from the same species can be used with the same labeled secondary antibody. Undesired reactions may occur if the secondary antibody cross-reacts with endogenous immunoglobulins in the specimen. This cross-reactivity can be eliminated by using secondary antibodies which are preabsorbed with immunoglobulin from the species from which the specimen is obtained.

Biotin-Streptavidin and Biotin-Avidin Detection
The binding of streptavidin or avidin to biotin is the most widely used amplification system for the staining of tissues and cells. In this method, unconjugated primary antibody binds to the antigen. Biotin-labeled antibodies then bind to the primary antibody. Addition of enzyme-labeled streptavidin or avidin results in the binding of multiple enzyme-labeled streptavidin or avidin conjugates to each biotinylated antibody (Figure 3.) The increase in enzyme indirectly bound results in signal amplification.

Figure 3:  ABC Method (avidin-biotin complex, left figure): Avidin- or streptavidin-biotin enzyme complex reacts with the biotinylated secondary antibody.  LSAB Method (Labeled streptavidin biotin, right figure): Enzyme-labelled (strept)avidin reacts with biotinylated secondary antibody.

Comparison of Streptavidin and Avidin
Streptavidin is prepared from Streptomyces avidinii, has a neutral isoelectric point (pI=approximately 7.0) and does not display the adverse binding characteristics attributed to avidin. Both proteins have the same high affinity for biotin (Kd=10-15
M-1). Avidin is isolated from egg white, has an alkaline isoelectric point (pI=10) and is glycosylated. This may result in nonspecific binding and potentially high background. Modification by succinylation or other chemical processes inhibits this phenomenon partially, but not completely.

Simultaneous Antigen Localization
The virtually irreversible binding of biotin by streptavidin provides a simple technique for multiple antigen detection when several different enzyme-labeled versions of streptavidin are available. After addition of a biotinylated link antibody and enzyme-labeled streptavidin, excess biotin is added to saturate unreacted biotin-binding sites. A second biotinylated antibody may then be added, followed by reaction with streptavidin labeled with a different enzyme. These additions can be continued as long as color reagents with sufficient contrast are available.

Labels
Common labels used to generate a signal and amplify the system in immunohistochemistry procedures include fluorochromes, enzymes, biotin and gold.

Fluorescent Labels
Antibodies labeled with fluorochromes to provide direct visualization are widely used in immunohistochemistry, flow cytometry and hybridoma screening. The variety of fluorochromes available with different emission spectra enables detection of two or more antigens on the same cell or tissue section. Fluorochrome-labeled antibodies offer sensitivity and high resolution; however, they have some disadvantages including the need for special equipment
(flow cytometer, fluorescence microscope) and results are not permanent. Regardless of these drawbacks, fluorescent techniques are often the method of choice because they
offer simple, rapid, highly sensitive detection.

Enzyme Labels
Antibodies labeled with enzymes are detected by reaction with chromogenic substrate which yields a colored precipitate that is visible by light microscopy. Enzyme-labeled antibodies are often preferred because they are inexpensive, highly sensitive and produce a permanent result.
Enzymes commonly used in immunohistochemical applications include peroxidase, alkaline phosphatase and ß-galactosidase. Horseradish peroxidase (HRP) is the most commonly used enzyme in immunohistochemistry. The fast catalytic rate of HRP allows for more product to be generated in a shorter amount of time; therefore, high sensitivity may be achieved quickly. Alkaline phosphatase (AP) exhibits a slower catalytic rate but the reaction is not self-limiting as is HRP. Reaction rates remain linear over longer periods of time; therefore, sensitivity may be improved by allowing the reaction to proceed for extended periods of time. ß-Galactosidase (ß-Gal) has an advantage over HRP and AP in that when it is used at optimal pH, interference from mammalian ß-Gal is eliminated. However, the use of ß-Gal is limited in application by the lack of substrate choices. For double and triple labeling,
any combination of the three enzymes may be used.

Gold Labels
Antibodies can also be conjugated to colloidal gold particles of different sizes. The use of gold can increase sensitivity and provide improved resolution over other methods when used on membranes or in cells and tissue. Antibodies conjugated with 5 nm colloidal gold particles are ideal for use in light microscopy. Antibodies conjugated with 40 nm gold particles are preferred for use in blotting applications.

Controlling the Procedure
Background staining is one of the most frequently encountered problems in immunohistochemistry. Some common causes of background staining are endogenous enzyme or biotin, hydrophobic binding between antibodies and tissue proteins, nonspecific cross-reactivity of antibody with antigen and non-specific binding of IgG by Fc receptors. In most cases, background can be minimized by proper blocking techniques and the choice of appropriate antibody.

Common Causes of Background

Blocking Endogenous Biotin
Biotin is a vitamin and coenzyme found in a variety of tissues such as liver and kidney. Nonspecific avidin or streptavidin binding is primarily due to endogenous biotin and is often present in avidin-biotin detection methods. Suppression
of endogenous avidin binding activity can be achieved by successive incubations of the section with 0.1% avidin and 0.01% biotin.

Hydrophobic Interaction
Hydrophobicity is a property shared by most proteins. Of the major serum proteins, the immunoglobulins are particularly hydrophobic. In tissue, proteins are rendered more hydrophobic by fixation with aldehyde-containing reagents such as formalin and gluteraldehyde. Factors in fixation procedures, such as time, temperature and pH during fixation, should be optimized to avoid excessive cross-linking of tissue proteins. The most widely used method to reduce background due to hydrophobic interaction is the use of blocking protein, such as bovine serum albumin (BSA) either prior to application of the primary antibody, as an additive in the antibody diluent or both. Hydrophobic binding between tissue proteins and immunoglobulins can also be minimized by lowering the ionic strength of the diluent buffer, by adding detergent to the diluent, or by raising the pH of the diluent.

Nonspecific Binding to Fc Receptors
Fc receptors are membrane glycoproteins with a high affinity for polymers and immune complexes of IgG. Blocking Fc reactive sites in tissues is often necessary to prevent nonspecific binding of antibodies to the tissue. Normal serum is most commonly used to block this reaction. Usually incubation of the tissue specimen with 10% normal serum (goat, rabbit, mouse, fish, etc.) is sufficient. An alternative is to use Fab or F(ab’)2 fragment antibodies, thereby removing the Fc binding
portion of the immunoglobulin.