Introduction
Immunohistochemistry is the localization of antigens in tissue sections by the use of labeled antibody as specific reagents through antigen-antibody interactions that are visualized by a marker such as fluorescent dye, enzyme, radioactive element or colloidal gold.
Albert H. Coons and his colleagues (Coons et al. 1941, 1955; Coons and Kaplan 1950) were the first to label antibodies with a fluorescent dye, and use it to identify antigens in tissue sections. With the expansion and development of immunohistochemistry technique, enzyme labels have been introduced such as peroxidase (Nakane and Pierce 1966; Avrameas and Uriel 1966) and alkaline phosphatase (Mason and Sammons 1978). Colloidal gold (Faulk and Taylor 1971) label has also been discovered and used to identify immunohistochemical reactions at both light and electron microscopy level. Other labels include radioactive elements, and the immunoreaction can be visualized by autoradiography.
Since immunohistochemistry involves specific antigen-antibody reaction, it has apparent advantage over traditionally used special enzyme staining techniques that identify only a limited number of proteins, enzymes and tissue structures. Therefore, immunohistochemistry has become a crucial technique and widely used in many medical research laboratories as well as clinical diagnostics.
There are numerous immunohistochemistry methods that may be used to localize antigens. The selection of a suitable method should be based on parameters such as the type of specimen under investigation and the degree of sensitivity required.
Tissue Preparation
Fixation:
Tissue preparation is the cornerstone of immunohistochemistry. To ensure the preservation of tissue architecture and cell morphology, prompt and adequate fixation is essential. However, inappropriate or prolonged fixation may significantly diminish the antibody binding capability.
There is no one universal fixative that is ideal for the demonstration of all antigens. However, in general, many antigens can be successfully demonstrated in formalin-fixed paraffin-embedded tissue sections. The discover and development of antigen retrieval techniques further enhanced the use of formalin as routine fixative for immunohistochemistry in many research laboratories.
For best results, vertebrate tissues (especially neuronal tissues) usually require fixation by transcardial perfusion for optimal tissue preservation. The most common fixatives used for immunohistochemistry are the followings:
a) 4% paraformaldehyde in 0.1M phosphate buffer
b) 2% paraformaldehyde with 0.2% picric acid in 0.1M phosphate buffer
c) PLP fixative: 4% paraformaldehyde, 0.2% periodate and 1.2% lysine in 0.1M phosphate buffer
d) 4% paraformaldehyde with 0.05% glutaraldehyde (TEM immunohistochemistry)
Some antigens will not survive even moderate amounts of aldehyde fixation. Under this condition, tissues should be rapidly fresh frozen in liquid nitrogen and cut with a cryostat without infiltrating with sucrose. The sections should be kept frozen at -20 C or lower until fixation with cold acetone or alcohol. After fixation, the sections can be processed using standard immunohistochemical staining protocols
Since its introduction, paraffin wax has remained the most widely used embedding medium for diagnostic histopathology in routine histological laboratories. Accordingly, the largest proportion of material for immunohistochemistry is formalin-fixed, paraffin-embedded. Paraffin sections produce satisfactory results for the demonstration of majority of tissue antigens with the use of antigen retrieval techniques.
Certain cell antigens do not survive routine fixation and paraffin embedding. So the use of frozen sections still remains essential for the demonstration of many antigens. However, the disadvantage of frozen sections includes poor morphology, poor resolution at higher magnifications, special storage needed, limited retrospective studies and cutting difficulty over paraffin sections.
Vibratome sections have some advantages when doing immunohistochemistry since the tissue is not processed through organic solvents or high heat, which can destroy the antigenicity. In addition, the morphology of tissue sections is not disrupted due to no freezing and thawing needed. Vibratome sections are often used for floating immunostaining, especially for pre-embedding EM immunohistochemistry. The disadvantage of vibratome sections is that the sectioning process is slow and difficult with soft and poorly fixed tissues. In addiction, the chatter marks or vibratome lines are often appeared in the sections.
Small blocks of tissue (less than 5 mm thick) can be processed as whole mounts. The advantage of whole mount preparations is that the results provide three dimensional information about the location of antigens without the need for reconstruction from sections. However, the major limitation of using whole mounts is antibody penetration may not be complete in the tissue, resulting in uneven staining or false negative staining. So Triton X-100 or saponin treatment are used routinely for whole mount immunohistochemistry to enhance penetration of the antibody.
Antigen Retrieval
The demonstration of many antigens can be significantly improved by the pretreatment with the antigen retrieval reagent that break the protein cross-links formed by formalin fixation and thereby uncover hidden antigenic sites. The techniques involved the application of heat for varying lengths of time to formalin-fixed, paraffin-embedded tissue sections in an aqueous solution (commonly referred to as the retrieval solution). This is called "Heat Induced Epitope Retrieval (HIER)". Another method uses enzyme digestion and is called "Proteolytic Induced Epitope Retrieval (PIER)".
Microwave Oven, Pressure Cooker and Steamer are the most commonly used heating devices. Other devices also include the use of autoclave and water bath. The heating length of 20 minutes appears to be the most satisfactory and the cooling usually takes about 20 minutes. Citrate buffer of pH6.0 is the most popularly used retrieval solution and is suitable for most of antibody applications. The TRIS-EDTA of pH9.0 and EDTA of pH8.0 are second most used retrieval solutions. Proteinase K is effective enzyme digestion reagent for membrane antigens such as Integrins, CD31, vWF, etc.
PIER methods (such as proteinase k, trypsin, chymotrypsin, pepsin, pronase and various other proteases) has also been reported for restoring immunoreactivity to tissue antigens with different degrees of success. However, the use of enzyme digestion method may destroy some epitopes and tissue morphology. Therefore the optimal enzyme concentration and incubation time need to be tested.
Combination of Heat Mediated and Proteolytic Enzyme Method is an alternative approach to unmask antigens if other methods did not work. It is especially useful when performing double or triple labeling of two or more antigens simultaneously.
Improving antibody penetration is also important for immunohistochemical staining of frozen and vibratome sections. Triton X-100 is by far the most popular detergent for improving antibody penetration for immunohistochemistry. However, it is not appropriate for the use of membrane antigens since triton X-100 destroy membranes. Some researchers prefer the freeze and thaw method for the improvement of antibody penetration. Sodium borohydride (1% in phosphate buffer) treatment is also widely used to unmask antigens, particularly in glutaraldehyde fixed tissue to reduce the glutaraldehyde linkages.
IHC Methods & Techniques
Blocking:
Background staining may be specific or non-specific. Inadequate or delayed fixation may give rise to false positive results due to the passive uptake of serum protein and diffusion of the antigen. Such false positives are common in the center of large tissue blocks or throughout tissues in which fixation was delayed.
Antibodies, specially polycolonal antibodies, are sometimes contaminated with other antibodies due to impure antigen used to immunize the host animal.
The main cause of non-specific background staining is non-immunological binding of the specific immune sera by hydrophobic and electrostatic forces to certain sites within tissue sections. This form of background staining is usually uniform and can be reduced by blocking those sites with normal serum.
Endogenous peroxidase activity is found in many tissues and can be detected by reacting fixed tissue sections with DAB substrate. The solution for eliminating endogenous peroxidase activity is by the pretreatment of the tissue section with hydrogen peroxide prior to incubation of primary antibody.
Many tissues also contain endogenous alkaline phosphatase (AP) activity and should be blocked by the pretreatment of the tissue section with levamisole if using AP as a label.
Some tissues such as liver and kidney have endogenous biotin. To avoid unwanted avidin binding to endogenous biotin if using biotin-avidin detection system, a step is necessary for these tissues by the pretreatment of unconjugated avidin which is then saturated with biotin.
Autofluorescence or natural fluorescence exists in some tissues and can cause background problems when fluorescent dyes are used in the experiments. The simplest test is to view the tissue sections with a fluorescence microscope before any antibody incubation. If autofluorescence is detected in the tissue sections, the best solution is to avoid use of fluorescent method but choose enzyme or other labeling methods.
Special controls must be run in order to test the protocol and for the specificity of the antibody being used.
Positive control is to test a protocol or procedure and make sure it works. It will be ideal to use the tissue of known positive as a control. If the positive control tissue showed negative staining, the protocol or procedure needs to be checked until a good positive staining is obtained.
Negative control is to test for the specificity of an antibody involved. First, no staining must be shown when omitting primary antibody or replacing an specific primary antibody with normal serum (must be the same species as primary antibody). This control is easy to achieve and can be used routinely in immunohistochemical staining.
Second, the staining must be inhibited by adsorption of a primary antibody with the purified antigen prior to its use, but not by adsorption with other related or unrelated antigens. This type of negative control is ideal and necessary in the characterization and evaluation of new antibodies but it is sometimes difficult to obtain the purified antigen, therefore it is rarely used routinely in immunohistochemical staining.
Direct Method:
Direct method is one step staining method, and involves a labeled antibody (i.e. FITC conjugated antiserum) reacting directly with the antigen in tissue sections. This technique utilizes only one antibody and the procedure is short and quick. However, it is insensitive due to little signal amplification and rarely used since the introduction of indirect method.
Indirect Method:
Indirect method involves an unlabeled primary antibody (first layer) which react with tissue antigen, and a labeled secondary antibody (second layer) react with primary antibody (Note: The secondary antibody must be against the IgG of the animal species in which the primary antibody has been raised). This method is more sensitive due to signal amplification through several secondary antibody reactions with different antigenic sites on the primary antibody. In addition, it is also economy since one labeled second layer antibody can be used with many first layer antibodies (raised from the same animal species) to different antigens.
The second layer antibody can be labeled with a fluorescent dye such as FITC, rhodamine or Texas red, and this is called indirect immunofluorescence method. The second layer antibody may be labeled with an enzyme such as peroxidase, alkaline phosphatase or glucose oxidase, and this is called indirect immunoenzyme method.
PAP Method (peroxidase anti-peroxidase method):
PAP method is a further development of the indirect technique and it involves a third layer which is a rabbit antibody to peroxidase, coupled with peroxidase to make a very stable peroxidase anti-peroxidase complex. The complex, composed of rabbit gaba-globulin and peroxidase, acts as a third layer antigen and becomes bound to the unconjugated goat anti-rabbit gaba-globulin of the second layer. The sensitivity is about 100 to 1000 times higher since the peroxidase molecule is not chemically conjugated to the anti IgG but immunologically bound, and loses none of its enzyme activity. It also allows for much higher dilution of the primary antibody, thus eliminating many of the unwanted antibodies and reducing non-specific background staining.
Avidin-Biotin Complex (ABC) Method:
ABC method is standard IHC method and one of widely used technique for immunhistochemical staining. Avidin, a large glycoprotein, can be labeled with peroxidase or fluorescein and has a very high affinity for biotin. Biotin, a low molecular weight vitamin, can be conjugated to a variety of biological molecules such as antibodies.
The technique involves three layers. The first layer is unlabeled primary antibody. The second layer is biotinylated secondary antibody. The third layer is a complex of avidin-biotin peroxidase. The peroxidase is then developed by the DAB or other substrate to produce different colorimetric end products.
Labeled StreptAvidin Biotin (LSAB) Method:
Streptavidin, derived from streptococcus avidini, is a recent innovation for substitution of avidin. The streptavidin molecule is uncharged relative to animal tissue, unlike avidin which has an isoelectric point of 10, and therefore electrostatic binding to tissue is eliminated. In addition, streptavidin does not contain carbohydrate groups which might bind to tissue lectins, resulting in some background staining.
LSAB is technically similar to standard ABC method. The first layer is unlabeled primary antibody. The second layer is biotinylated secondary antibody. The third layer is Enzyme-Streptavidin conjugates (HRP-Streptavidin or AP-Streptavidin) to replace the complex of avidin-biotin peroxidase. The enzyme is then visualized by application of the substrate chromogen solutions to produce different colorimetric end products. The third layer can also be Fluorescent dye-Streptavidin such as FITC-Streptavidin if fluorescence labeling is preferred.
A recent report suggests that LSAB method is about 5 to 10 times more sensitive than standard ABC method.
1) EnVision Systems are based on dextran polymer technology. This unique chemistry permits binding of a large number of enzyme molecules (horseradish peroxidase or alkaline phosphatase) to a secondary antibody via the dextran backbone. The benefits are many, including increased sensitivity, minimized non-specific background staining and a reduction in the total number of assay steps as compared to conventional techniques. The simple protocol is i) Application of primary antibody; ii) Application of enzyme labeled polymer; iii) Application of the substrate chromogen. EnVision+ was developed after EnVision to provide increased sensitivity.
2) ImmPRESS polymerized reporter enzyme staining system is based on a new method of polymerizing enzymes and attaching these polymers to antibodies. The novel approach employed to form enzyme "micropolymers" avoids the intrinsic shortcomings of using large dextrans or other macromolecules as backbones. Attaching a unique "micropolyer with a high density of very active enzyme to a secondary antibody generates a reagent that overcomes steric interference and provides enhanced accessibility to its target. The result is outstanding sensitivity, signal intensity, low background staining, and reduced non-specific binding. The simple protocol is i) Application of primary antibody; ii) Application of enzyme labeled polymer; iii) Application of the substrate chromogen.
1) CSA Systems use Tyramide Signal Amplification. It is ideal for the following applications: i) Detecting small quantities of antigen; ii) Enhancing performance of low affinity mouse and rabbit antibodies; iii) Enabling compatibility of certain "tough" mouse and rabbit antibodies with paraffin embedded tissue sections. The simple protocol is as follows:
1. Application of primary antibody.
2. Application of biotinylated linking antibody.
3. Application of the Tyramide Amplification Reagent.
4. Application of Streptavidin-HRP.
5. Application of the substrate chromogen
2) CSA II - Biotin-free Tyramide Signal Amplification System is a highly sensitive immunohistochemical (IHC) staining procedure incorporating a signal amplification method based on the peroxidase-catalyzed deposition of a fluorescein-labelled phenolic compound, followed by a secondary reaction with a peroxidase-conjugated anti-fluorescein. In the procedure, a mouse primary antibody is first detected with a peroxidase-conjugated secondary antibody. The next step utilizes the bound peroxidase to catalyze oxidation of a fluorescein-conjugated phenol (fluorescyl-tyramide) which then precipitates onto the specimen. The procedure is continued with detection of the bound fluorescein by a peroxidase-conjugated anti-fluorescein. Staining is completed using diaminobenzidine/hydrogen peroxide as chromogen/substrate, and can be observed with a light microscope. In comparison to standard immunohistochemical methods, such as labelled streptavidin biotin (LSAB) or avidin-biotin complexes (ABC), tyramide amplification methods have been reported to be many fold more sensitive. The CSA II System is a simplified version of the extremely sensitive Catalyzed Signal Amplification System (code K1500) that utilizes biotinyl-tyramide. The highly sensitive CSA II System allows for the detection of very small quantities of target protein, as well as for the use of low affinity antibodies. This reagent system utilizes fluorescyl-tyramide, rather than biotinyl-tyramide, and does not contain avidin/biotin reagents, thus eliminating potential background staining due to reactivity with endogenous biotin.
Principles of Procedure: The specimens are first incubated with Peroxidase Block for five minutes to quench endogenous peroxidase activity. The specimens are then incubated for five minutes with a protein block to suppress nonspecific binding of subsequent reagents, followed by a 15-minute incubation with an appropriately characterized and diluted mouse primary antibody or negative control reagent (user provided). This is followed by sequential 15-minute incubations with anti-mouse immunoglobulins-HRP, fluorescyl-tyramide hydrogen peroxide (amplification reagent) and anti-fluorescein-HRP. Staining is completed by a five-minute incubation with 3,3' diaminobenzidine tetrahydrochloride (DAB)/hydrogen peroxide, which results in a brown precipitate at the antigen site.
Immunohistochemistry Sensitivity Chart:
| Standard Sensitivity | Moderate Sensitivity | Most Sensitivity |
| ABC Method, PAP Method, LSAB Method | EnVision, EnVision+, ImmPRESS | CSA Method, CSA II Method |
Multiple Labeling
It is often useful to be able to stain for two or more antigens in one common tissue section. This can be achieved by immunofluorescence method using different fluorescent dyes. Multiple staining can also be done with peroxidase conjugated antibodies developed with different chromogen substrates to produce the end products of different colors. There are three basic approaches in planning multiple staining: parallel, sequential and adjacent. In addition, the antibody dilution and condition are also important factors to be considered. Finally, appropriate color combination is also crucial since improper color combination may produce poor result and fail to demonstrate multiple antigens in the same section. For best result, the careful design and test of multiple staining protocols are necessary.
Immuno-Electron Microscopy
Electron microscopic (EM) immunohistichemical techniques can be divided into two groups: Those where the immunostaining takes place prior to resin embedding are referred to as pre-embedding. Those methods where the immunolabeling is undertaken after resin embedding are known as post-embedding.
The choice of whether to apply pre- or post-embedding method to the detection of an antigen in any particular location will depend to a large extent upon the distribution and liability of the antigen and the characteristics of the primary antibody. Before starting immuno-EM labeling, a test for the characteristics and dilution of the primary antibody should be performed at light microcopy level.
Several recently developed methods rely on labeling with colloidal gold particles. These methods were originally introduced for electron microscopy (Faulk and Taylor 1971) as the gold particles are easily visible under the electron microscope, but they are also useful for light microscopy.
Since gold particles can be made in different size from 5 to 30 nm, it is possible to carry out multiple staining at the electron microscopic level, most easily by direct labeling of several first layer antibodies with different sized particles. The indirect techniques can also be used in double or triple labeling by parallel approach if the primary antibodies are from different species, and by sequential approach if the primary antibodies are from same species.