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Immunostaining Techniques

Immunohistochemistry / Immunocytochemistry

Immunohistochemistry/immunocytochemistry is the demonstration of a tissue or cellular constituent in situ by detecting specific antibody-antigen interactions where the antibody has been tagged with a visible label. The visual marker may be a fluorescent dye, colloidal metal, hapten, radioactive marker or an enzyme. Experimental samples ranging from fresh frozen cells and sections to heavily fixed Paraffin- or Resin-embedded whole tissue samples have been used. Despite the diversity of techniques now available to the researcher, ideally, maximal signal strength along with minimal background or non-specific staining are required to give optimal antigen demonstration.

Central to these techniques is the use of an antibody to link a cellular antigen specifically to a stain that can be readily visualized with a microscope. Detection of antigens in tissues is known as Immunohistochemistry, while detection in cultured cells is generally referred to as Immunocytochemistry. For both, there is a wide range of specimen source, antigen availability, antigen-antibody affinity, antibody type, and detection enhancement methods. Thus optimal conditions for immunohistochemical or immunocytochemical detection must be determined for each individual situation, dependent on the above variables. As for all procedures, reference should be made to individual product data sheets and published literature. Also, the Internet today contains a tremendous amount of useful information on Immunohistochemistry/ Immunocytochemistry. Quick searches through google.com or similar search engines are highly recommended.

Since the protocols provided in this section are general, it is highly recommended that the researcher review the methodology and variations in IHC protocols; Immunocytochemical Methods and Protocols (second edition), edited by Lorette C. Javois, from Methods in Molecular Medicine, volume 115, Humana Press, 1999 (ISBN 0-89603-570-0).

Specimen Preparation

  • Fixatives
  • Section Type

Fixatives

Fixatives are needed to preserve cells and tissues in a reproducible and life-like manner. An ideal fixative should:

  1. Preserve the tissue and cells without any shrinking or swelling and without distorting or dissolving cellular constituents.
  2. Prevent putrefaction by bacteria and prevent autolysis by cathepsin containing cells.
  3. Stabilize and protect tissues and cells against the detrimental effects of subsequent processing and staining procedures.

To achieve this, tissue blocks, tissue sections, cell cultures or smears are either immersed in a fixative fluid, or in cases where whole animal systems are studied, the animal is perfused with fixative via its circulatory system. In the case of cells in culture, cell preparations are either submerged in fixative or simply air-dried. Fixatives stabilize cells and tissues thereby protecting them from the rigors of subsequent processing and staining techniques. No fixative meets all of the above criteria and some compromise is necessary. The choice of fixative will depend upon the type of specimen and the components to be demonstrated.

For immunological studies, careful consideration of the fixation protocol is especially imperative to ensure the adequate preservation of the specimen and target antigens. Tissues have differing protein content and structural arrangement, thus they have a variable ability to retain their structure without significant fixation. Incorrect specimen preparation can block or impede antigen labeling in tissue and cells. Unfortunately, the methods that are best for the preservation of tissue structure do so by altering proteins, thereby masking some epitopes, and sometimes preventing the detection of the desired target protein. In cases of failure it is important to experiment with multiple different fixatives and antigen retrieval methods prior to "giving up" on a specific stain.

Immunohistochemistry Process

Fixatives may work by several means: formation of crosslinkages (e.g., aldehydes such as glutaraldehyde or formalin); protein denaturation by coagulation (e.g., acetone and methanol); or a combination of the above. Fixation strengths and times must be optimized so that antigens and cellular structures can be retained and epitope masking is minimal. Requirements for fixation can vary widely between tissues. For example when using antibodies to probe for neurotransmitter substances, most tissues must be either immersion fixed with a mixture of glutaraldehyde and paraformaldehyde, or with paraformaldehyde alone. Both acetone and methanol (precooled to -20°C) have been used successfully as fixatives for frozen tissue in other instances

The most widely used fixatives are formalin based. The three most commonly employed fixatives for general use are neutral buffered formalin, formal saline, or 10% formalin in dH2O.

Formalin, like other aldehyde fixatives, forms cross linking methylene bridges and Schiff bases between basic amino acid (lysine) residues of proteins. This cross linking stabilizes the proteins in situ, which is the basis of fixation. Formaldehyde produces mild cross linkages when compared to other aldehyde fixatives such as glutaraldehyde. In addition to the choice of fixative, other important factors include fixation time, temperature and pH. Fixation time will depend upon the size of the specimen. In order to achieve adequate and consistent fixation it is essential that tissue specimens be sliced to a thickness of 3 mm or less.

Immunocytochemistry

Fixation of cultured cells for immunocytochemistry also requires careful consideration; in general, fixation strengths and times are considerably shorter for cells than on the thicker, structurally complex tissue sections. For immunocytochemistry, sample preparation essentially entails fixing the target cells to the slide. Depending upon the needs of the experiment, cells can be simply harvested and "dropped" on to a slide, fixed and air-dried, as is often done with clinical or diagnostic studies where speed and a simple "yes/no" answer is all that is needed. Alternatively, where information on the structural location within the cell is needed, cells are often cultured directly on prepared slides or coverslip material, which are then simply washed and fixed in place prior to staining. Consulting published literature relating to the tissue/ proteins of interest is well worth the time invested. See Appendix for recipes of common fixatives.

The next consideration for immunological staining is the type of section to use. For immunohistochemistry, the common options are fixed or unfixed cryostat (frozen) sections, fixed "wet" or vibritome sections, or fixed, paraffin-embedded sections. The choice of section is determined by a number of issues, including the time and skill of the investigator. Because of the ease of use, fixed frozen sections are often quickest and easiest to use. However, because of their superior fidelity, clarity, and preservational properties, fixed paraffin-embedded tissues have become the ultimate standard of immunohistochemisty in histology and pathology, and anytime where archiving of immunohistochemical information is required.

Section Type

  • Cryostat (frozen) sections
  • Paraffin (wax) sections
  • Resin (plastic) sections

Cryostat (Frozen) Sections

Cryostat (Frozen) Sections
Rabbit anti-α-internexin (Cat. No. AB5354) and Mouse anti-NF-L (Cat. No. MAB1615) staining of cultured rat neurona. Mature neurona are green. (NF-L positive) and neuronal progenitor cells are red (α-internexin positive).

There are two types of Cryostat sections: (1) Fresh, or unfixed sections where quickly frozen (snap frozen) tissues are first cut, then either air-dried or fixed prior to staining; (2) or fixed frozen tissue, where the tissue is first fixed then cryoprotected with sucrose or other stabilizer (to stabilize the tissue cell structure) prior to freezing and sectioning. The advantages of frozen sections are that they allow excellent antigen preservation, they are typically faster to perform, and they offer flexibility, since any fixative can be used, thereby facilitating the optimization of fixative for each antigen. However frozen sections give less morphological detail and resolution than other methods.

The freezing procedure is extremely important, if carried out too slowly it will result in the formation of ice crystal artifact, which may make histological interpretation very difficult. For optimum results tissue needs to be sliced thinly (maximum thickness 3 mm) and rapidly frozen in liquid nitrogen. Frozen sections are cut at a thickness of 5 m using a cryostat. The sections are dried overnight at 37°C before fixation and subsequent staining or storage. For subsequent immunohistochemical staining the sections are fixed in acetone for 20 minutes.

It is important to dry the sections thoroughly, in order to optimize tissue morphology by reducing the effects of chromatolysis and membrane breakdown, which is particularly evident with techniques involving long incubations in aqueous solutions. The drying of sections overnight at 37°C does not affect section antigenicity. Frozen tissue can be stored in liquid nitrogen or at -70°C for many years without affecting antigenicity. The tissue should be wrapped in aluminum foil or immersed in embedding medium to prevent the tissue from drying out. Similarly, frozen sections can be wrapped in aluminum foil and stored at -20°C for a year or more without loss of antigenicity.

Paraffin Sections

The largest proportion of samples used in immunostaining are embedded in paraffin because it provides for excellent morphological detail and resolution. Modern "paraffin" is typically a mixture of paraffin wax and resin. It is an excellent embedding medium because it can be heated to liquid state, and dissolved by xylene for infiltrating the tissue, and then relatively quickly turned to a solid state again for maximum structural support during sectioning. Typically, small blocks (10x0x3 mm) of tissue are fixed for up to 24 hrs. The most common fixatives used in paraffin sections are formalin-based. These fixatives are well tolerated by the tissues and achieve good penetration. See Appendix for recipes of common fixatives. The blocks are then infiltrated and embedded with paraffin and 3-10 m sections are cut in ribbons, floated in a 45°C water bath, and dried onto slides. Thicker sections may now be used due to computer enhanced imaging equipment like confocal microscopes but may make antibody penetration more difficult. Once mounted, the slides can be stored indefinitely until immunostaining is required; then the paraffin must be removed from the tissue to allow the water-based buffers and antibodies to penetrate.

Paraffin Sections
Rabbit anti-Indolamine 2, 3-dioxygenase (Cat. No. AB5968) staining of a lymph node from a human patient with malignant melanoma.Malignant cells are stained red.

Resin Sections

Embedding tissues in resin offers several advantages over frozen or paraffin sections, thinner sections may be used with improved morphology and less tissue shrinkage. Also, calcified material may be sectioned without prior decalcification and infiltration of acrylic resins and polymerization can take place at low temperatures. The use of a modified methyl methacrylate (MMA) was developed by: Hand N, Morrell K. (1990). Immunocytochemistry on plastic sections for light microscopy: a novel post-embedding procedure. Proceedings of the Royal Microscopical Society 24:A54-55.

Thie resin, which is soluble, consists of the monomer methyl methacrylate with dibutyl phthalate as the plasticiser, N-N-dimethylaniline as an accelerator and benzoyl peroxide as the catalyst. This method produces comparable immunostaining to that obtained on paraffin sections, but has the added advantage of giving improved morphology and resolution.

Antigen Retrieval

  • Enzymatic Digestion
  • Heat mediated
  • Microwave
  • Autoclaving or Pressure Cooking

To facilitate the immunological reaction of antibodies with antigens in fixed tissue or cells (less common), it may be necessary to unmask or "retrieve" the antigens through pretreatment of the specimens. There are many forms of antigen retrieval (sometimes called antigen recovery), and different antigens and different antibodies will require different antigen retrieval methods. Tissue sections are unique to each experimental condition. Differences in the type and duration of fixation, together with variations in tissue processing, reagents and the manner by which sections are dried after cutting are important considerations. There are great variations in the way these processes are performed in research laboratories. The lack of standardization of the fundamental processes of immunohistochemical methods culminates in the production of a unique preparation. Therefore, one particular method of antigen retrieval may produce optimal staining for one condition, but not another.

Antigen retrieval has been shown to increase reactivity of the majority of antigens in tissues. The use of antigen retrieval in immunocytochemistry is less common, however depending upon the particular antibody/antigen combination it can be performed on cell preparations, although the length of time and intensity is typically much less than for tissue.

The vast majority of antigen retrieval studies have been applied to formalin fixed material. When aldehyde-based fixatives are used (e.g., formalin), inter- and intra-molecular cross-links are produced with certain structural proteins, which are responsible for the masking of tissue antigens. This adverse effect has been thought to be due to the formation of methylene bridges between reactive sites on tissue proteins. These reactive sites include primary amines, amide groups, thiols, alcoholic hydroxyl groups and cyclic aromatic rings. The degree of masking of the antigenic sites depends upon the length of time of fixation, temperature, concentration of fixative and the availability of other nearby proteins able to undergo cross-linkages. In order to "unmask" these antigenic sites a range of antigen retrieval techniques are available.

Antigen retrieval includes a variety of methods by which the availability of the antigen for interaction with a specific antibody is maximized. The most common techniques are enzymatic digestion or heat induced epitope retrieval (HIER) through microwave irradiation, autoclaving or pressure cooking.

Proteolytic Enzyme Digestion

This technique involves dewaxing, rehydrating, and rinsing the specimen in running water. The specimen is then equilibrated with the appropriate buffer, and incubated with a proteolytic enzyme at 37°C, or at room temperature. Enzymes used include pronase (0.05% (w/v) in PBS), trypsin (0.05% (w/v) in PBS with 0.1% CaCl2) and pepsin (0.05% (w/v) in 2 normal Hcl). The conditions of concentration, time and temperature must be controlled, so that the enzymes can break some of the bonds formed during fixation, uncovering antigenic sites, but the antigen should not be digested completely. Digestion time is extremely important and is proportional to the specimen fixation time. There is a very fine balance between over and under digestion. For example, trypsin is optimally active at 37°C and at pH 7.8. The reaction rate is improved by the addition of the co-enzyme calcium chloride (0.1%). Trypsin only remains active for about 30 minutes; therefore if the incubation time exceeds this, the working solution must be replaced. The enzymatic activity is stopped by placing the specimen in cold buffer (4°C) prior to processing with antibody. These methods should be considered for some antigens/tissues. (Shi, S-R et al. (1993). J. Histochemistry & Cytochemistry 41:1599-1604) althouth not all antigens require proteolytic digestion. Indeed, proteolytic enzymes can abolish the reactivity of some antigens. (Pileri, S. et al. (1997). J. Pathology 183:116-123). Furthermore, care must be taken to avoid creating "false" antigenic sites, as some antigens may be altered or destroyed by trypsinization. In some instances immunostaining may be impaired or completely removed following trypsinization. Although sometimes necessary for particular antibody protocols, the inconsistant nature of proteolytic digestion treatments have caused many researchers to favor heat mediated antigen retrieval methods.

Heat Mediated Antigen Retrieval

The rationale behind these heat pretreatment methods is unclear and several theories have been postulated. One theory is that heavy metal salts act as a protein precipitant, forming insoluble complexes with polypeptides and that protein precipitating fixatives frequently display better preservation of antigens than do cross- linking aldehyde fixatives. Another theory is that during formalin fixation inter- and intra-molecular cross methylene bridges form linkages and weak Schiff bases. These cross linkages alter the protein conformation of the antigen such that a specific antibody may not recognize it. It is postulated that heat mediated antigen retrieval removes the weaker Schiff bases but does not affect the methylene bridges so that the resulting protein conformation is intermediate between fixed and unfixed.

Antigens masked during routine fixation and processing can be revealed by using the following high temperature, heat mediated antigen retrieval techniques; microwave oven irradiation, combined microwave oven irradiation and proteolytic enzyme digestion, pressure cooker heating, autoclave heating, water bath heating, Steamer heating, or high temperature incubator. Microwave and autoclave antigen retrieval are discussed further below.

Microwave Irradiation

Microwave irradiation of formalin-fixed, paraffin- embedded specimens in buffer has been found to markedly enhance the retrieval of antigens. During this procedure the energy provided helps break some of the bonds formed during fixation, thus increasing the number of positive cells available, and the intensity of reactions, although the exact mechanism is unclear.

It is important to monitor the sections during the microwaving process, to prevent damage and drying. Consistency of conditions between experiments, including buffer volumes, irradiation times, and microwave unit used, will result in less variability in staining results. The number of samples that can be treated by microwave irradiation at one time is limited. Typically specimens in some buffer (see below) are heated either at full or partial power for a few minutes. Periodically the heating is stopped and liquid is replenished. After a set time, the solution containing the slides is allowed to cool to room temperature slowly, then the slides are rinsed in PBS and used for staining. Reference: Shi SR, Key ME, Kalra KL. (1991), Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39(6):741-8.

Microwave Irradiation
Mouse anti-Synaptophysin (Cat No. MAB 329). Localization of synaptophysin (red) and Myelin Basic Protein (green) in human hippocampus (paraffin-embedded section).

Autoclaving or Pressure Cooking

In order to standardize the procedure, it is important to start with standard volumes of preheated solutions. After adding the specimens to the boiling retrieval solution, the autoclave or pressure cooker should be brought to full pressure as quickly as possible and the heating times measured exactly from this point. At the end of the heating time (usually 1 to 2 minutes) the pressure should be released. As soon as possible the hot buffer should be flushed out with cold water. (Sections should not be allowed to dry.) The specimens should then be washed in buffer.

Although the most critical feature of both microwaving and autoclaving is probably the heating of the tissues, the pH and composition of the solutions used are also important in the unmasking of antigenic sites. Studies have found no significant difference between microwave and autoclave treatment, but there are significant differences based on the solutions used. Some of the buffer solutions commonly used are 0. 01 M citrate buffer (pH 6.0), 0.1 M Tris-HCl (pH 8.0) and 1 mM EDTA (pH 8.0), with citrate buffer used most commonly. It should be noted that many more specimens can be treated at any one time using an autoclave or pressure cooker than using a microwave oven. However, preservation of the cytological detail may be slightly inferior in sections that undergo pressure cooking.

A more mild procedure that can be used on many tissues is a simple incubation in citric acid buffer, pH 3.0 (2.1 grams Citric Acid added to 400 mL of ddH20. Adjust to pH 3.0 with Acetic acid if above 3.0, or NaOH if below 3.0, make up to 1 L final volume with ddH20) for 30 minutes, at 37°C after blocking but prior to primary antibody addition. Rinse slide in PBS or TBS pH 7.4 prior to staining.

Antibody Staining

The primary antibody may be directly labeled with an enzyme (such as horseradish peroxidase or alkaline phosphatase) or afluorophore (such as FITC or rhodamine), or unlabeled, with detection by a labeled secondary antibody or more complex detection system. If a secondary antibody is used, it must be generated against the immunoglobulins of the primary antibody source, e.g., if the primary antibody is raised in rabbit, then the secondary antibody could be goat anti-rabbit. The optimal titer of both the primary and secondary antibody should be determined for each batch.

The proper working dilutions for every antibody must be optimized for the system in which it is being employed. The same system does not always work for every antibody. Antibodies are like children, and each is different. The product data sheets may be used as a guide for dilution series starting points. The optimal antibody dilution will be that which gives the strongest specific antigen staining with the lowest non-specific background. As with other controlled experiments, it is advisable to change only one experimental variable at a time. After determining the optimum titer/dilution of the primary antibody, the secondary antibody dilution can be optimized.

For staining of tissue sections, it is customary to incubate with 25-50 L of diluted antibody - the volume used must be sufficient to completely cover the tissue, and to ensure the tissue will not dry out during incubation. Incubation times may range from 30-90 minutes at 37°C, from one to six hours at room temperature, or overnight at 4°C. Incubation times should be optimized empirically for each antibody/antigen combination.

Millipore's IHC Select® Manual Staining System (Cat. No. MSS001) offers a convenient platform for quality antibody staining. This compact device allows simultaneous IHC staining of 2 to 40 microscope slides, and offers the convenience of standardized staining with the efficiency of capillary gap technology to reduce reagent volume and minimize waste.

Antibody Detection

  • Enzyme-Mediated
  • Fluorescence
  • Signal Amplification

Antibody Detection
GFAP (Cat. No. IHC2079-6 Clone GA5) staining of human brain. Tissue pretreated with citrate buffer, pH 6.0, prediluted polyclonal antibody, IHC Selects detection with HRP-DAB. Glial cells stain strongly (brown).

Two of the most commonly used detection methods are fluorescence and colorimetric (enzyme mediated) detection. With the advent of electron microscopy, detection of antigens by antibodies that contain large gold particles is often used, and these may also be visualized at the light microscopic level as well, but their use is quite rare today, outside of electron microscopy. Described below are the common antibody detection methods for light microscopy.

Enzyme-Mediated Detection

When choosing a substrate for conversion by an enzyme, one should select a substrate which yields a precipitating product. Examples of commonly-used substrates are listed below.

Substrate Abbrev- iation Final Color Soluble in Alcohol (for counter- stain) Comments
Diaminobenzidine DAB Brown No 3,3'-diaminobenzidine produces a brown end product which is highly insoluble in alcohol and other organic solvents. Oxidation of DAB also causes polymerization. DAB has the ability to react with osmium tetroxide, and thus is very useful in electromicroscopy as well as traditional immunohistochemistry sections.
Diaminobenzidine with nickel enhancement DAB/Nickel Gray /Black No Produces a more intense stain which is resistant to alcohol and provides better contrast, up to 40 times more sensitive than DAB without enhancement
3-Amino-9-ethylcarbazole AEC Red/Brown Yes AEC produces a red/brown reaction product Brown and is widely used for immunohistochemical staining. Slide specimens processed with AEC must not be immersed in alcohol or alcoholic solutions (e.g., Harris' hematoxylin). Instead, an aqueous counterstain and mounting medium should be used. AEC is also susceptible to further oxidation when exposed to light and thus it will fade overtime. Dark storage and brief light viewing are recommended.
4-Chloro-1-naphthol 4-CN Blue/Gray Yes 4-chloro-1-naphthol (CN) precipitates as a blue Gray end product. Because CN is soluble in alcohol and other organic solvents, the slides must not be dehydrated, exposed to alcoholic counterstains, or coverslipped with mounting media containing organic solvents. Unlike DAB, CN tends to diffuse from the site of precipitation, thus it is not usually recommended for Immunohistochemistry but can be used for Western blotting
Naphthol AS B1 phosphate/fast Red TR NABP/FR Red Yes Napthol AS-acts as the substrate for alkaline phosphate/fast red TR phosphatase, and the Fast Red chromogen precipitates at the enzymatic sites producing a vibrant red/pink color. Precipitate is soluable in alcohol, thus aqueous counterstain and mounting medium should be used
Naphthol AS MX phosphate/fast Red TR NAMP/FR Red Yes Napthol AS- acts as the substrate for alkaline phosphatase, and the fast red chromogen precipitates at the enzymatic sites producing a vibrant red/pink color. Precipitate is soluable in alcohol thus aqueous counterstain and mounting medium should be used
Naphthol AS B1 phosphate/new fuschin NABP/NF Red/ Violet Yes Napthol AS- acts as the substrate for alkaline phosphatase, and the new Fuchsin chromogen precipitates at the enzymatic sites producing a vibrant red/violet color. Precipitate is soluable in alcohol, thus aqueous counterstain and mounting medium should be used.
Bromochloroindolyphos- phate/Nitro Blue Tetrazolium BCIP/NBT Purple No 5-bromo,4-chloro,3-indolylphosphate (BCIP)/ nitroblue tetrazolium (NBT) substrate is a commonly used substrate chromogen. BCIP acts as the substrate for alkaline phosphatase, and the NBT enhances the purplish-brown color of the precipitate. BCIP/NBT is compatible with organic solvents so it can be used with alcohol based counterstains including Nuclear Fast Red or Methylene-Green.

Fluorescence

A molecule that fluoresces can be attached to the antibody for detection using UV light. Examples are Fluorescein, Rhodamine, Texas Red®, Cy3 and Cy5. In selecting fluorochromes, one is limited by the available microscope filter sets. Most filter sets are best matched with rhodamine or fluorescein. Texas Red® may also be used with a rhodamine filter set.

Many mounting media contain "anti-fading" solutions, such as DABCO, which will prolong the viewing time of the sample. Millipore offers a variety of fluorescence mounting fluids and counterstain solutions including our basic fluorescent mounting fluid (Cat. No. 5013) and enhanced counterstaining fluid containing nuclear stains such as DAPI and PI (Cat. No. S7113, S7112).

Signal Amplification

Signal amplification techniques greatly enhance the sensitivity of immunohistochemical and immunocytochemical methods. The signal amplification methods may be used in conjugation with either of the above detection techniques. Signals may be amplified by using poly-conjugated secondary antibodies (i.e. Millipore catalog numbers AP340P-AP342A series), or Avidin-Biotin interactions or other commerciallyavailable amplifiers (i.e tyramide catalyzed systems), which increase the signal to antibody ratio. When signal amplification is used to amplify the specific signal, however, one should be aware that non-specific signals may also become amplified. Thorough washing and proper antibody titration is especially important in this case.

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