Immunohistochemical methods for studying mononuclear phagocytes in tissue sections

ELSEVIER

JOURNALOF IMMUNOLOGICAL METHODS Journal of ImmunologicalMethods 174 (1994) 133-154

Immunohistochemical methods for studying mononuclear phagocytes in tissue sections Francois Plenat a,*, Yves Martinet b, Nadine Martinet c, Jean-Michel Vignaud

a

a Laboratoire d'Anatomie Pathologique, CHU de Nancy, H3pital Central, 31 rue Lionnois, 54000 Nancy, France b Service de Pneumologie, CHU de Nancy, H3pital de Brabois, Avenue du Morvan, 54500 Vandoeuvre Les Nancy, France c INSERM, Unit~ 14 de Physiopathologie Respiratoire, C.O. n ° 10, 54511 Vandoeuvre Les Nancy, France

Abstract

This paper reviews the main immunohistochemical techniques that can be used for studying mononuclear phagocytes in tissue sections. Keywords: Immunohistology; Monocyte; Macrophage

1. Introduction

Immunocytochemical techniques permit cellular or tissue constituents (antigens) to be localized and identified in situ in cell or tissue preparations. Immunohistochemistry is now a rich and complex field (in terms both of the different techniques available and the large n u m b e r of different molecular entities which are detectable).

Abbreviations: AP, alkaline phosphatase; APAAP, phosphatase-antiphosphatase; APTES, aminopropyl-triethoxysilane; BCIP, 5-bromo-4-chloro-3-indolyl-phosphate; DAB, 3,3'-diaminobenzidine; FITC, fluorescein isothiocyanate; HRP, horseradish peroxidase; NBT, nitro blue tetrazolium; OCT, optimal cutting temperature embedding medium; PAP, peroxidase-antiperoxidase; PFA, paraformaldehyde; RT, room temperature; TRITC, tetramethyl rhodamine isothiocyanate. * Corresponding author. Tel.: (33) 83.85.13.51; Fax: (33) 83.85.11.49.

The aim of this p a p e r is to give a detailed description of the immunohistochemical procedures that can be used for studying mononuclear phagocytes in tissue sections. These methods can be applied to cytological preparations but this aspect will not be specifically dealt with. It is essential to be familiar with the fundamental principles of immunohistochemical methods so that the technique can be more fully understood and effectively applied. Accordingly, we will initially give the background of the techniques. For m o r e complete reviews see Bullock and Petruz (1983, 1985), Polak and Van N o r d e n (1983), H a r low and Lane (1988).

2. Choice of antibodies

Tissue staining techniques d e m a n d that an antibody binds to a specific antigen in the presence of high concentrations of other macromolecules,

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and this places exceptional demands on antibody specificity. Antibody preparations that are satisfactory for immunoblotting or immunoprecipitation may show spurious cross-reactions in tissue staining. 2.1. Polyclonal antisera A good polyclonal serum will contain multiple antibodies reacting with a broad range of epitopes on the antigen. Although steric competition may prevent large numbers of antibodies from binding to the same antigen molecule, several antibodies will often bind, and a strong signal is expected. One major drawback of polyclonal antisera is that, even when they have been raised against highly purified antigens, they may contain unexpected antibodies against other constituents present in tissue. Because these antibodies will normally not be the major activities in the serum, their binding can often be reduced below the level of detection by careful titration. Contaminating activities that persist at optimal working dilutions can frequently be removed by preabsorbing the serum with suitable acetone powder or by affinity purification before use. A further drawback of polyclonal antibodies is that, although a particular batch of antiserum may be of high specificity, maintenance of the same quality in subsequent batches may be difficult to ensure.

clonal antibodies do not give satisfactory results in staining fixed paraffin sections, presumably because the epitope is hidden within a cell structure or is denatured during fixation. Many laboratories have now started to develop monoclonal antibodies that will perform well on these paraffin-embedded tissues by immunizing mice with formalin-fixed antigens and screening fusions using tissue sections. The theoretical possibility that a monoclonal antibody will detect a conformational epitope present on two related molecules is a risk which in practice has turned out to be a very rare event. A far more common situation is that a molecule which is initially believed to be specific for a particular cell type proves, on further study, to be more widely distributed. This is commonly observed for white-cell associated antigens. Examples of this phenomenon include the expression of the MAX. 3 antigen by alveolar macrophages, megakaryocytes and platelets (Andreesen et al., 1986), and the CD4 antigen present on helper-inducer T-cells and macrophages (Barclay et al., 1992). The problem of quality control, referred to above in the context of polyclonal antisera, is most often easily overcome when monoclonal antibodies are used, since hybridoma cell lines can produce an unlimited amount of identical antibodies.

3. Choice of label

2.2. Monoclonal antibodies Monoclonal antibodies are the products of individual clones of lymphocytes. They will usually react only with a single determinant on any individual molecule (unless the molecule is multimeric or contains repeating antigenic sequences). In consequence, it might be predicted that monoclonal antibodies will always lead to lower intensity of immunohistochemical labeling than can be achieved with polyclonal antisera. Fortunately this theorical objection does not appear to be supported by practical experience. Monoclonal antibodies often work exceptionally well in tissue staining techniques, where their purity and specificity yield low backgrounds over a wide range of antibody concentrations. However, many mono-

The labels used to visualize an antibody fall into three main classes: fluorescent, enzymatic and gold with or without silver enhancement. It is also possible to use a radioactive label followed by autoradiography. This is an uncommon immunohistochemical procedure and the technique will not be discussed here. Several manufacturers now produce a range of conjugated detector molecules of high quality. Producing conjugates in the laboratory is not recommended when commercial reagents are available. 3.1. Fluorescent label An ideal fluorescent label has a high quantum yield, good separation between the wavelengths

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of excitation and emission, a wavelength of maximal absorption close to a strong line from a mercury lamp (used for fluorescence microscopy) and an emission wavelength suitable for photographic film and the human eye. The original definitive description of the immunofluorescent method was based on the use of fluoresceinlabeled conjugates. FITC continues to be the primary fluorochrome of choice due to its superior performance when compared with alternative compounds that have been proposed over the past 35 years. Fluorescein conjugates show an absorption maximum of 495 nm and have a strong fluorescence emission which is apple-green, a color rarely encountered in mammalian tissues as autofluorescence and which also corresponds to a region of high retinal sensitivity. The principal alternative to fluorescein is TRITC. Conjugates of this dye show an absorption maximum of 555 nm and an orange fluorescence giving a good contrast with fluorescein. The intensity of the label is less than with FITC though the persistence of the signal is longer. It is widely employed in dual-fluorescence studies. An alternative orange/red-emitting fluorochrome, Texas red, has been proposed. It offers the high emission of FITC in conjunction with the better persistence of TRITC. It is excited and emits in the same range as TRITC, although the emitted signal has a slightly more orange characteristic. Recently, B phycoerythrin, a fluorescent protein found in red algae, has been used. Unlike rhodamine this dye shows orange fluorescence on excitation by light of similar wavelengths to that used to excite fluorescein, which makes it easily distinguishable from FITC. However, the major use of this fluorochrome has been in flow cytometry. Transmitted light systems have now been superseded by the incident system in which the specimen is illuminated through the objective lens so that the actual surface viewed is exposed directly to the fluorescence-exciting radiation. The system depends on the use of beam-splitting dichroic mirrors which reflect the excitation light onto the specimen and transmit back to the oculars the light of wavelength corresponding to the fluorescence emission. Furthermore epi-illumination permits an easy changeover between fluorescence

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microscopy and transmitted light microscopy since the substage illumination remains readily available. This is very useful for a number of applications, such as immunofluorescence in combination with phase-contrast microscopy.

3.2. Enzymatic labels When an enzyme is used as a label, it is visualized by means of a reaction which gives an insoluble end-product. An ideal enzyme has a low molecular mass (for easy attachment of the reporter molecules to proteins and optimal tissue penetration of enzyme-protein complexes into the tissues) and a high turnover number (to give a high yield of product), is absent from the normal tissues and can be used to give a product that is insoluble in water, dehydrating and clearing agents (so that the coverslips can be mounted conventionally). Most often, the enzyme is covalently coupled to an antibody, and a variety of techniques are well established for this purpose. Chemical coupling procedures have, however, several shortcomings, particularly as they adversely affect antibody as well as enzyme reactivity. Therefore, unconjugated enzyme-antibody methods, such as the peroxidase-antiperoxidase or the phosphatase-antiphosphatase methods have been developed in which the marker enzyme is fixed to the site of the antigen by several steps of antibody binding instead of chemical coupling. The most widely used enzyme in these procedures is HRP. This molecule is small (M r approximately 40000) by comparison to an antibody molecule, relatively easy and inexpensive to purify, and enzymatically stable. It fulfills most of the above criteria except that it is found in some normal tissues and cell lineages. Staining of endogenous peroxidase may, indeed, present problems of interpretation in tissues containing many granulocytes (both neutrophils and eosinophils). Immunoperoxidase methods are thus best avoided for studying blood and bone marrow preparations. Resident macrophages have peroxidase activity in the rough endoplasmic reticulum, the nuclear envelope, and - regularly - in the Golgi apparatus. Peroxidase activity is also present in the cytoplasmic granules of exudate, and

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exudate-resident macrophages. This, of course, is more of a problem in cryostat sections than in paraffin sections in which much of the native enzyme activity is destroyed during the processing, but even there it may be intrusive. In these cells, however, inhibition of endogenous activity can be achieved by treating the sections with a solution containing H 2 0 2 and NaN 3 after incubation with the primary antibody (vide infra). A positive histochemical reaction is also given by the hemoglobin of erythrocytes though this is not considered to be due to truly enzymatic catalysis. The chromogen used most frequently in the HRP methods is DAB with H202, yielding a browncolored precipitate, insoluble in organic solvents, and contrasting sharply with hematoxylin or methyl green counterstains. The reaction product with DAB can be intensified or its color changed using various salts of heavy metals. These intensification reagents, e.g., 0.12% (w/v) osmium tetroxide, 0.5% (w/v) copper sulfate, 1% (w/v) cobalt chloride, or 1% (w/v) nickel ammonium sulfate may be used by adding them to the DAB solution or as a post-incubation procedure. De Jong et al. (1985) have systematically studied most of these procedures and concluded that a DABcobalt or DAB-nickel system yielded the best enhancement. Darkening of the reaction product, however, may be associated with an unwanted increase of background. In some laboratories, 4-chloro-l-naphthol or 3-amino-9-ethylcarbazol are used as alternative substrates, since the possibility has been raised that DAB may be carcinogenic. These histoenzymatic methods are less sensitive and satisfactory because the end-products of the reactions do not withstand alcohol and solvent-based mounting media. During the incubation, all media for visualizing peroxidase activity become slightly turbid a n d / o r change in color. However, background staining does not appear if the incubation times are kept short (10-20 min). Longer periods of incubation in chromogen solution did not result in a more marked reaction product but in visible background staining. This is especially true for the DAB-metal ions protocol. Histochemical specificity is obtained as long as this oxidation occurs only at sites that contain peroxidase activity. Strong light also acts as an

oxidizer and as a consequence leads to nonspecific precipitates of chromogens. Protection of sections from light in peroxidase histochemistry results in lower levels of background staining. Calf alkaline phosphatase (AP) is an alternative enzyme to HRP. Although this enzyme is of a higher molecular weight (M r approximately 115000) and less easily purified than HRP, in practice it produces results which are equivalent to or better than those obtained using HRP. From a practical point of view, there is no need to block endogenous enzyme activity prior to incubation with the primary or secondary antibody as this may be achieved by the addition of levamisole to the substrate medium. This is possible since the calf intestinal enzyme in the immunodetection system is resistant to the effects of levamisole. In certain tissues (for example placenta), however, it may be difficult to reduce the endogenous background activity. A large number of different colored substrates can be used to detect the enzyme. Commonly used substrates contain naphthol phosphate (naphthol AS-MX or naphthol AS-BI with a diazonium or hexazonium salt). The perfect azoic compound has not yet been found, but hexazotized New Fuchsin is one of the best available. Its vivid red reaction product is insoluble in organic solvents. Frequently used diazonium salts are Fast red TR (which gives a red precipitate, soluble in alcohol and xylene) and Fast blue RR (which gives a blue precipitate soluble in organic solvents). Using these dyes a slight increase of background staining of sections is found with time. This light yellow-orange background staining is due to the reaction of diazonium or hexazonium salts with tissue constituents such as the side chains of amino acids. However, it does not interfere with the color of the specific precipitate. An alternative method for the localization of AP activity was introduced by McGadey (1970). It is mainly used for immunoblotting and the detection of hybrids in in situ hybridization procedures. The principle of the method is that the substrate BCIP is hydrolysed at alkaline pH with a concomitant release of hydrogen which can reduce the tetrazolium salt NBT. The BCIP/NBT substrate generates an intense black-purple precipitate at the site of

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enzyme binding. The substrate solution is stable, thus allowing development of the reaction for up to 24 h. This method is the most sensitive but the visual quality of the results is often inferior to that obtained with azoic substrates. Furthermore when the tissue sections have not been pretreated as for in situ hybridization, background staining may occur.

to control. The final staining result is often 'dirty' and difficult to interpret. Furthermore, positive artifacts may occur due to non-catalytic formation of metallic silver, especially in dense structures such as red blood ceils and nuclei.

3.3. Colloidal gold

Increasing sensitivity for antigen detection is continually being sought. The reasons for this include not only the detection of smaller amounts of antigen, but also the ability to use antibodies at higher dilutions. In addition, a detection method that is suitable for identifying a particular antigen in unfixed frozen tissue may not be ideally suited for detecting the same antigen after fixation, with greater amplification being most often necessary in the latter situation. This gives rise to a plethora of different detection systems. As in many other immunological techniques, detection methods can be divided into two groups: the direct and indirect methods. The choice of the method depends on the experimental design.

Typically either an anti-IgG serum or another detector protein is bound to gold particles of known diameter and used to identify the sites of attachment of the primary antibody. Small colloidal gold particles (1-5 nm diameter) yield the most sensitive results. The labeling procedures yield a permanent, non-bleaching staining. Sections can be dehydrated, mounted in commonly used embedding media and stored for further examination. Structures on cells or in tissues to which immunoglobulin-gold complexes have been bound appeared red when examined by brightfield illumination. However, as the tissue-bound quantity of gold-coated protein is small, only faint staining is usually observed in the light microscope. De Mey (1983) has found that polarized light epi-illumination microscopy is more sensitive than bright-field illumination for the detection of tissue-bound gold particles. Stained structures appear as bright yellow spots on a dark, almost structureless, background. It is also possible to convert even optically unnoticed deposit into black staining by treating the sections with a physical silver developer. Colloidal gold catalyses the reduction of silver (I) ions to metallic silver generating dense and optically visible deposits from weak gold labeling. The intensity of staining needs to be monitored by frequent microscopic examination of tissues. Such silver intensification can also be applied to HRP-labeled antibodies, since the DAB-polymer also possesses catalytic properties. The use of a dark room for development, whilst desirable, is not obligatory provided the sections are protected from unnecessary exposure to light during the procedure. Our experience with silver intensification has not been very satisfactory in that the reaction is often difficult

4. Methods of application

4.1. Direct methods

In the direct method, the antigen-specific antibody is purified, labeled, and used to bind directly to the antigen. This method is the simplest (one step) for detecting an antigen in a tissue. Primary antibodies labeled with fluorescent labels or chromogenic enzymes are available for many human antigens and have their major application in the field of hematology. Direct labeled antibody methods are rapidly performed, and relatively economical, but are less sensitive than the indirect methods. Indeed, even the primary enzyme-labeled antibodies commercialized by DAKO under the name of EPOS that permit an enhanced one-step staining are, in our hands, less sensitive than the classical multi-step methods. They are thus most often utilized with frozen tissues. The reduced sensitivity is not only due to the lower degree of amplification of the immunohistochemical method, when compared with indirect methods, but also to modifications of the primary antibody. Direct labeled antibody meth-

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ods are also accompanied by a higher level of background than that seen with other immunohistochemical procedures, and require a new labeling step for every antibody to be studied.

4.2. Indirect methods In the indirect method, the antigen-specific antibody is unlabeled. Its binding to the antigen is detected by a secondary reagent.

4.2.1. Indirect labeled antibody methods In the two-step indirect method, an unlabeled primary antibody bound to its specific antigen in the tissue is revealed with a labeled secondary antibody raised against the primary antibody in another animal species. This method gives good results on fixed or unfixed frozen sections but is not usually sensitive enough to work on paraffinembedded tissue. Adding a third layer (threestage indirect method) will yield additional amplification. For example, if, in a direct labeling experiment, one labeled mouse monoclonal antibody molecule can bind to the antigen, using an indirect method five labeled rabbit anti-mouse immunoglobulin antibodies are able to bind, and in a triple-layer experiment 25 labeled goat antirabbit immunoglobulin antibodies will bind, thereby increasing the original signal 25 times. 4.2.2. Methods involving the use of enzyme~antienzyme complexes In these methods, complexes of enzyme and anti-enzyme antibodies are prepared. If the two molecules are added at nearly equal molecular ratios, multimeric interactions occur quickly, thereby allowing the formation of large enzyme/ anti-enzyme complexes. These complexes are prepared in slight enzyme excess to avoid precipitation. These large complexes are linked to the primary antibody using a bridging anti-immunoglobulin antibody. The enzyme/anti-enzyme complexes must be prepared with antibodies of the same species and isotype as those of the primary antibody. The second layer antibody must be in excess with respect to the first layer so that there is competition between the antibody molecules for the bound first-layer primary antibody. Thus

only one combining site of each of the secondlayer antibody molecules is occupied by the primary antibody, now acting as an immunoglobulin antigen, and the second site is free to combine with the enzyme-anti-enzyme complexes, another immunoglobulin antigen. The most common examples of this method are the PAP (Sternberger et al., 1970) and APAAP complexes (Cordell et al., 1977). Since monoclonal antibodies recognize only one specific type of antigenic determinant, monoclonal PAP or APAAP complexes have a lower molecular weight than polyclonal PAP and APAAP complexes which facilitates penetration. These complexes can be purchased in their large aggregated forms. These unlabeled antibody techniques tend to give low background and are of a comparable or better sensitivity than indirect procedures. Furthermore, their sensitivity may be further increased by repeating the bridging anti-Ig antibody and PAP or APAAP steps.

4.2.3. The biotin-avidin (streptavidin) methods A further important category of immunohistochemical procedures takes advantage of the high affinity of the reaction between the egg-white protein avidin (or streptavidin, its bacterial relative from Streptomyces avidinii) and the vitamin biotin (K a = 1015). Avidin has a relative molecular weight of 66000 and a p I of 10.5. Under physiological conditions its carbohydrate moieties can bind to certain lectins and owing to its positive charge, it also binds to negatively charged biomolecules. These intrinsic properties significantly reduce the specificity of avidin as well as its general utility. In contrast, streptavidin (M r = 60 000) does not have carbohydrate moieties and has a neutral pI. Thus avidin has been almost completely superseded by streptavidin for the detection of biotinylated molecules including biotinylated antibodies. Streptavidin may be easily gold labeled or conjugated with fluorochromes or enzymes. Three different methods are commonly used in biotin/streptravidin detection systems: (1) streptavidin is used as a bridge between the primary target (biotinylated primary or more frequently secondary antibody) and the detector (biotinylated enzymes); (2) a preformed complex of streptavidin and biotinylated enzyme is used to

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detect the biotinyl residues of labeled antibodies; and (3) a conjugate that is prepared by chemically cross-linking enzymes to streptavidin is used for the detection. In our hands, all three methods have a similar sensitivity and the third is the easiest to perform. In practice, the biotin-streptavidin method is no more sensitive than the other procedures described above. This may be explained by the fact that a chain is only as strong as its weakest link, that the very high affinity of this binding is less important for the intensity of the final reaction than the affinity of the second antibody for the primary antibody, or of the primary antibody for tissue antigen. Although generally applicable to any type of section, there is a potential for an increase in background when the streptavidin-biotin methods are used as a detection system in staining frozen sections. Presumably, this is due to the presence in some samples of high concentrations of endogenous biotin which is normally destroyed in the course of the paraffin-embedding procedure. It is thus advisable not to use biotin-based methodology on frozen sections.

5. Tissue processing Before deciding how to handle the tissue, the effect of any processing on the antigen of interest must be considered. Theoretically, antigenic substances in cells and tissues that have been prepared for immunohistochemistry should be preserved in their natural form, in the exact sites in which they are located. This is most often true but it must be realized that the native conformation and structure of antigens may be partially lost during the procedures used for their preparation. Consequently such antibodies are raised against denatured or modified antigens. This may explain why some antibodies work surprisingly well on paraffin-embedded sections whereas poor or negative results are obtained on cryostat unfixed or briefly fixed sections. It must also be realized that each tissue and antigen is different. Because their biochemical composition is highly variable, they may respond differently to fixatives and treatments designed to unmask antigens and

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may also be subject to variable levels of adventitious binding of the antibody. It is thus somewhat unrealistic to expect that a single procedure will perform for all tissues. Therefore, when using new antibodies the optimum conditions for fixation and processing can only be established empirically, by first testing the antibodies on tissues known to contain the antigens. It should be noted that the various epitopes on an antigen might be affected differently by a fixative which explains why polyclonal antisera usually work well on paraffin-embedded fixed sections. If several monoclonal antibodies recognizing the same antigen are available, each should be optimized separately. 5.1. The choice of the optimum regimen o f fixation

The choice of the procedure used greatly influences the immunohistochemical results subsequently obtained. Fixation entails conversion of soluble native constituents into insoluble derivatives, by treatment with some fixative agents. Such agents mainly include chemically reactive compounds and both organic and inorganic solvents. The fixation process not only alters retained biopolymers but also influences morphology and changes various physical characteristics of cells and tissues (such as permeability or electric charge), which in turn influences the staining outcomes. Since antigens display different physicochemical properties (biochemical composition, isoelectric point, solubility and conformation, etc.) that will affect their reactivity with fixatives, it is not possible to make general rules covering all systems but the following considerations apply. (1) Cross-linking reagents such as glutaraldehyde or formaldehyde with or without additional chemicals such as picric acid or dichromate have a considerable inhibiting effect on many antigens. However, a mixture of periodate, lysine and paraformaldehyde (PLP) provides a low-denaturing fixative, combining good immunohistochemical reactions with acceptable morphology. PLP has been used in studies of several surface determinants, especially in lymphoid cells, and has been compared with other common fixatives particularly in regard to overall efficacy. Antigen

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masking by aldehyde fixatives not only depends on the antigen concentration but also on the environmental proteins surrounding the antigen which may prevent antigen-antibody interaction by steric hindrance. This may be partially reversed in some instances by treating the sections before immunostaining with proteolytic enzymes a n d / o r by microwave heating the sections. Many of the markers detected by monoclonal antibodies on paraffin sections of samples fixed by crosslinking reagents are carbohydrates. This may be because carbohydrates are preserved after routine formalin fixation and many laboratories use fixed sections for screening the reactivity of monoclonal antibodies. In addition, carbohydrates are sterically restricted structures that are strongly immunogenic. (2) Many antigens are resistant to organic solvents such as methanol, ethanol, acetone, or chloroform. (3) Ethanol/acetic acid (3/1, v/v) is the best fixative for measuring the incorporation into DNA of a thymidine analogue such as bromo- or iododeoxyuridine (Duprez et al., 1989). The method of fixation can also dramatically influence morphology. Perfusion fixation is recommended where possible. Immersion fixation must, of course, suffice for human material. Finally, it is essential to fix tissues with the minimum of delay in order to avoid autolytie degradation of antigens. The choice of the optimum regimen is thus achieved by juggling with the fixative identity (formaldehyde or acetone and so on) and the fixation conditions (which pH, temperature, concentration, osmolarity, time, perfusion or immersion?). 5.2. The use of frozen sections During routine embedding procedures, temperature-dependent destruction of antigenic determinants may occur. Generally speaking, antibodies to extracellular and surface antigens can only be applied reliably to frozen sections. Tissue staining studies on mammalian tissue are, therefore, often carried out on frozen sections of unfixed or fixed tissues. The need for frozen sections has several obvious disavantages over the

use of conventional paraffin-processed tissues such as the difficulty of achieving good morphological preservation, the diffusion or loss of soluble antigens resulting in poor localization and reduced sensitivity, and the need of committed personnel, space, and facilities in order to maintain a properly supervised tissue 'bank'. In the majority of cases, the tissue is frozen without prior fixation. Tissue sections are then subjected to brief fixation using gentle fixatives. In this laboratory we would select as a first choice 100% acetone, 100% chloroform, 80% ethanol or 4% paraformaldehyde in PBS at - 4 ° C or at RT for 2-10 min. If all fixatives appear to destroy the antigen, the staining must be carried out without prior fixation; this will give sections with poor morphology. A specimen must be frozen in such a way as to minimize the formation of ice crystals, which can produce abundant conspicuous holes in the tissue. This artifact is usually seen in large specimens that have been allowed to freeze in a deepfreeze cabinet or dipped directly into liquid nitrogen. We have found, as have others, that improper freezing and thawing artifacts are more of a serious problem than delays in tissue freezing. Suitable techniques include immersion of specimens which should not be more than 2 mm thick in isopentane cooled to its freezing point by liquid nitrogen, or placing samples on a metal block that has previously been brought to the temperature of either liquid nitrogen or dry ice and acetone mixture. Before freezing, specimens are most often immersed in chemically inactive hydrophilic substances of low molecular weight in order to control the plasticity of frozen tissues (the so-called optimal cutting temperature embedding media (OCT)). In addition, this treatment also reduces the size of the ice crystals formed. Damage due to ice can also be greatly reduced by cryoprotection, in which the specimen is first equilibrated with a solution of a cryoprotective compound in physiological buffer (e.g., glycerol, dimethyl sulfoxide, or sucrose). Infusion is carried out at 4°C, for up to 12 h, depending on the size of the tissue pieces to be studied. It should finally be noted that long-term storage of tissue blocks at low temperatures has a desiccat-

F. Plenatet al. /Journal of ImmunologicalMethods 174 (1994) 133-154 ing effect and so the storage system should take this into consideration. 5.3. The use of paraffin-embedded sections If the antigen survives one or more of the fixatives, the tissue is fixed in the optimum fixative and the effects of embedding tissue on the preservation of antigenicity can be studied. Antibodies to cytoplasmic antigens (intermediate filaments, lysozyme and a-chymotrypsin, some markers of macrophage differentiation) frequently work well in formalin-fixed paraffin-embedded sections. Monoclonal anti-BUdR antibody binding to IUdR-substituted DNA can be easily carried out in tissue sections of ethanol-acetic acid fixed paraffin-embedded tissues. During routine embedding procedures, however, temperaturedependent destruction of antigenic determinants may occur which can, sometimes, be prevented by using paraffins with lower melting point instead of the conventional paraffin (45°C as opposed to 58°C). 5.4. Effects of decalcifying solutions on immunoreactivity Specimens of calcified tissues are normally decalcified before embedding in paraffin wax. Matthews and Mason (1984) suggested that neutral 0.5 M EDTA, 10% (w/v) formic acid, and 10% (v/v) acetic acid produce little modulation of the immunoreactivity with monoclonal and polyclonal antibodies, even after as long as 7 weeks of decalcification. However, with decalcifying agents containing mineral acids unreliable results are obtained. Protease treatment (see below) is often necessary to maximize immunostaining results with all decalcifying reagents.

6. Miscellaneous practical considerations 6.1. Reduction of non-specific binding of immunoglobulins Primary antibodies may attach themselves to highly charged tissular sites, e.g., collagen or nu-

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clei. As the ionic bonds between the primary antibodies and the tissue are of low affinity, rinsing under conditions of high salt concentration (0.5 M) after immunoreaction will prevent the formation of these non-specific bonds. It is also often possible to reduce this background staining by pre-incubating the slides with a solution of protein in buffer. The alternatives include bovine serum albumin (BSA) (3% (w/v) in buffer), fetal bovine serum (30% (v/v) in buffer), normal serum from the animal in which the secondary antibody was produced (1/3 to neat) or human serum at the same concentration. These reagents may also be used to dilute antibodies and are sometimes included in washing solutions. Normal sera must be decomplemented at 56°C for 1 h. In our experience, the most efficient way for blocking nonspecific background staining with protein is to pre-incubate the sections in a solution of nonfat dried milk. It is easy to use and compatible with many of the detection systems in common use. There is only one circumstance under which nonfat dried milk should not be used: when tissue sections are probed for an antigen that may be present in milk. An even better signal-to-noise ratio can be obtained by addition to the solution of sodium heparinate and a non-ionic surfactant such as Triton X-100, Tween 20 or Nonidet P40. Having incubated the blocking solution on the tissue sections for 10-30 rain, it should be carefully poured away, leaving a film over the whole section. Care must be taken not to leave to much solution on the slides as this will cause dilution of the primary antibody. Although unnecessary with most monoclonal antibody reagents which usually give clean immunostaining, a 'blocking step' is advisable for the majority of immunostains performed with polyclonal antisera. Tissue-bound aldehyde groups which bind all proteins strongly and non-specifically are a major source of trouble when the fixative contains glutaraldehyde. The aldehydes should be converted to unreactive hydroxyls by reduction with sodium borohydride, before attempting any immunohistochemical procedure. Glycine may also be used to quench the free aldehyde groups in tissues, the aldehyde groups reacting with the free amino group of glycine. Immunologists with experience of work-

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ing with living cells are well aware that immunoglobulins may bind via its Fc portion to specific receptors on the surface of human cells. However, when staining fixed tissue sections, this appears not to present a major problem, probably because fixation destroys receptor activity. Background staining due to the reactivity of the second step antibody with endogenous mouse immunoglobulins may be a serious problem when mouse tissues are stained with mouse monoclonal antibodies. The magnitude of this problem will depend on the presence of endogenous immunoglobulins or B lymphocytes. If background staining of this origin is a problem, it is worth biotinylating or fluoresceinylating the primary antibodies and detecting the tissue-fixed primary antibodies with a streptavidin-based methodology or an indirect multi-step immunohistochemical technique using a polyclonal anti-fluorescein antiserum as secondary antibody.

6.2. Assisting antibodies to penetrate the tissue and reversing some of the effects on tissues of cross-linking fixatives Antigen may be masked by other molecules that obstruct access to the antibody molecules. The most obvious barriers to penetration are lipoprotein membranes and cytoplasmic matrices that have been tightly cross-linked by an aldehyde containing fixative. To improve penetration of antibodies, detergents such as 0.1-1% (v/v) Triton X-100, Tween 20 or Nonidet P40 may be incorporated in a pre-incubation soaking in buffer. They may also be added to the antibody solutions and buffer rinses. Non-ionic surfactants are used because they do not impart electric charge, which might cause non-specific antibody binding to the specimen. Tissue antigens which have been sequestrated during fixation and processing can also often be revealed by treating tissue sections with proteolytic enzymes, e.g., trypsin, pronase, pepsin, ficin, or bromelain. This works well, for example, on formalin-fixed, paraffin-embedded tissues with some antibodies to CD68. Most of our experience has been with trypsin. It must be prepared fresh and in a suitable solvent. Most antigens are proteins or pep-

tides. It is, therefore, important not to use prolonged proteolytic digestion. A pH well removed from the optimum for the enzyme (as in the experimental conditions described below) helps to moderate the destructive action of the enzyme under variable conditions. The duration of proteolytic digestion will depend upon a number of factors: time in fixative, nature of fixative and antigen under consideration. Hence, for every antibody used, it will be necessary to perform basic trials to ascertain the optimum conditions with regard to digestion for routinely processed tissues. Attention to the type of trypsin used is also essential and the results may differ accordingly. Another very efficient method for recovering some masked antigens is based on microwave heating of tissue sections to temperature up to 100°C in the presence of buffer solutions containing or not heavy metals salts (Shi et al., 1991). The dramatic enhancing effect of this microwave treatment on the recovery of many antigens is particularly intriguing in view of the presumed deleterious effects of high temperatures on protein antigens. Finally, thorough washing of sections from formalin-fixed paraffin-embedded material before immunostaining is begun may also reverse some of the effects of cross-linking by formalin fixation and make more antigen available to the antibody.

7. Multiple staining The number of antigens which can be detected in human tissue is increasing steadily year by year and this has led to a growing need for methods which will enable the relative distribution patterns of pairs of antigens to be visualized in tissue sections. Basically there are three main ways to obtain information about the possible co-localization of two antigens: (1) the comparison of individual immunohistochemical staining patterns on serial sections, (2) the use of double immunofluorescence methods, (3) the application of immunoenzyme double-staining techniques or the combination of an immunoenzyme technique with an immunogold staining procedure. Immunofluorescent or immunoenzyme techniques on serial sec-

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tions give reliable results but 'cell to cell' comparison within homogeneous structured tissues such as lymphoid tissue is difficult because of the small size of the cells and, in fluorescence microscopy, the lack of tissue landmarks. Furthermore, the serial sections must be very thin to avoid small ceils not to be present in both sections. Technically, this may be a problem for cryostat and paraffin sections. The results of immunoenzyme double-staining techniques on single slides are satisfactory if the two antigens are located on separate cells or are found in separate cellular compartments. If not, it is difficult to unequivocally distinguish a single from a doubly-labeled cell. In this case, fluorescent labels must be used. New double-bandpass filter sets (e.g., filter set 23 from Zeiss) have been tailored to the fluorochromes FITC and TRITC or Texas red. Thus, the documentation of specimens marked with these fluorochromes now no longer requires any change of filter. Photography of both fluorescence markers is highly accurate since image deplacement is absolutely impossible. Demonstration of multiple antigens on the same section by enzyme immunohistochemistry may be achieved using one type of enzyme with different substrates, or different enzymes. Particular attention has to be given to the selection of appropriate enzymatic substrates. A successful double-staining experiment depends on the presence of two highly contrasting colored precipitates after histochemical development of the tracer enzyme activities. The process of selection of the best method to adopt for an immunoenzyme double staining was recently described in detail by Van der Loos at al. (1993). Double staining may also be achieved by a combination of immunogold-silver staining with an immunoenzymatic procedure. When the primary antibodies have been raised in different species and the second labeled immunological reagents are not cross-reactive, the two methods may be carried out simultaneously with a mixture of two reagents in each layer. Simultaneous detection of two antigens on the same section can also be successfully carried out with antibodies or streptavidin labeled with two different enzymes. Sequential application of two immunoenzymatic sandwiches is, however, often easier to perform.

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The controls must always include single staining with each primary antibody.

8. Controls and interpretations of results All these immunophenotyping techniques require appropriate positive and negative controls. The reliability of reagents and antibodies must be continually monitored to avoid erroneous results and the expenditure of extensive and time-consuming technology. For each antibody, a block of tissue known to contain the antigen should be selected and a large number of sections cut. One of these should be included in every run in order to monitor the strength of the staining reaction. Each run should also include an experimental section on which the first antibody has been omitted. This checks for non-specific staining by the reagents used to detect the primary antibody. If an enzyme stain is being used, a section which is developed for color only is included in order to monitor endogenous enzyme activity. These controls do not check for non-specific staining by the primary antibody. When using a polyclonal antiserum, an aliquot of serum can be absorbed with the original antigen. This should reduce the specific and non-specific stain. It is frequently assumed, when a cell gives a positive immunocytochemical reaction in a tissue section, that it must have synthesized the antigen which it contains. However, it is now clear that diffusion and artifactual uptake of extracellular antigens may occur, probably during tissue processing, with the result that the cytoplasm of a cell may contain an antigen acquired from its environment. Proteins which are normally present in the serum in high concentrations, e.g., immunoglobulins and transferrin, are particularly likely to produce such artifacts. When a cell in a tissue section gives a negative immunohistochemical reaction, the obvious interpretation is that it does not contain the antigen in question. However, a variety of mechanisms may account for a cell which does contain an antigen apparently yielding a negative immunohistochemical reaction. Denaturation of antigens has already been referred to. The level of antigen in

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the cell may also be too low to be detected. This may be because only small amounts of the antigen are synthesized by the cell. An example would be a growth factor receptor present at low density on the cell surface. Alternatively, a molecule may be synthesized in large amounts but be exported rapidly from the cell, and hence never accumulate at a detectable concentration. Nota bene: very low levels of antigen may sometimes be successfully detected using a pool of monoclonal antibodies to the antigen, provided the antibodies in the pool can bind to it non-competitively. This increase in signal can mean the difference between successful detection and failure.

9. Experimental methods The methods described here are in use in the authors' laboratory. Many satisfactory variations are to be found in the literature and can be introduced as long as certain guidelines are followed.

9.1. Buffers Buffers are used to prepare some of the fixatives, during the staining procedures to rinse and wash slides, as diluents for the antibodies, and to prepare enzymatic incubation media. The addition of physiological saline to the buffer solution gives it a higher salt concentration, thus effectively reducing non-specific background staining. It is a routine procedure in this laboratory to dilute antibodies in buffers containing 0.5% BSA, and also to add 0.5% BSA to the washing buffers. The most commonly used buffers are the following. 0.1 M phosphate-buffered saline. Dissolve 8.0 g of NaC1, 0.2 g of KCI, 21.72 g of Na2HPO 4- 7 H 2 0 , and 2.6 g of K H 2 P O 4 in 800 ml of distilled water. Adjust the pH to 7.4 with NaOH or HCI. Bring volume up to 1 liter with distilled water. Mix well. Store at RT. Phosphate ions may inhibit AP. Thus, in immunohistochemical procedures using AP as the label, TBS is the preferred buffer. Tris-buffered saline. Add 800 ml distilled water,

6.61 g Tris-HC1, 0.97 g Tris base, 8.77 g NaCl. Bring volume up to 1 liter. Mix well. Nota bene: The amino groups of Tris react with aldehydes and so Tris buffers are not used with formaldehyde- and glutaraldehyde-based fixatives. Both of these buffers may be purchased commercially. It is important to note, however, that some manufacturers add sodium azide (NaN 3) to the buffer formulation as an antibacterial agent and this will inhibit the binding of HRP to the substrate in the visualization procedure. 0.01 M citrate buffer p H 6.0. (1) Stock solutions: (A) 0.1 M citric acid (21.01 g C 6 H 8 0 7 • H 2 0 in 1000 ml distilled water; (B) 0.1 M sodium citrate (29.41 g C 6 H 5 0 7 N a 3 . H 2 0 in 1000 ml distilled water. (2) Working solution: 9 ml of A + 41 ml of B, diluted to a final volume of 500 ml.

9.2. Fixatives Neutral buffered formalin. Dissolve 4.0 g N a H 2 P O 4 - H 2 0 and 6.5 g Na2HPO 4 in distilled water. Add 100 ml of commercial formalin (3740% HCHO) and make up to 1 liter with water. Buffered formaldehyde. To prepare a methanol-free solution of paraformaldehyde, weigh out a quantity of paraformaldehyde that will make a 4% solution and add it to a volume of water equal to slightly less than 2 / 3 of the desired final volume of fixative. Cover. Transfer to a fume hood and heat to 60°C with magnetic stirring. To aid solubilisation, add a few drops of 2 N NaOH. The solution should become clear fairly rapidly. When paraformaldehyde has completely dissolved, let the solution cool to RT. Bring the pH of solution to 7.2 with HCI and make up to the final volume with water. Store at 4°C (12 h maximum). Caution: Formaldehyde is a potential carcinogen and may cause allergic reactions. Buffered glutaraldehyde. Buffered solutions are used at concentrations from 0.25 to 5%, either alone or in conjunction with other aldehydes. Glutaraldehyde is available as an EM grade redistilled 25% aqueous solution, the pH of which should lie between 4 and 5. It can be stored for 3-4 months at 4°C. When stored at RT it polymerizes and has a slightly yellow color. Such

F. Plenatet al. /Journal of Immunological Methods 174 (1994) 133-154 solutions should be discarded. Glutaraldehyde, in fact, is essentially used for immunohistochemistry at the electron microscopic level. Bouin's fixative. 800 ml saturated alcoholic picric acid, 150 ml formalin (37-40% HCHO), 50 ml glacial acetic acid. This mixture can be kept indefinitely, but its chemical composition changes considerably with time. Caution: picric acid is dangerously explosive when dry and therefore must be stored under water. Periodate-lysine-paraforrnaldehyde. Prepare a 3.6% (w/v) solution of paraformaldehyde by dissolving 2 g in 0.14 M NaH2PO4, 0.11 M NaOH at 70°C. Filter, cool and bring the pH of solution to 7.4. Bring the pH of a 0.2 M solution of lysine monohydrochloride to 7.4 by the addition of 0.1 M Na2HPO 4. Dilute to 0.1 M lysine by the addition of 0.1 M phosphate buffer, pH 7.4. Just before use, mix 1 part of paraformaldehyde solution to 3 parts lysine solution and add sodium paraperiodate to a concentration of 10 mM. Ethanol Various percentages of ethanol at 4°C or RT for a predetermined time. 3 / 1 (v / v) ethanol/acetic acid. 120 ml 90% ethanol, 40 ml glacial acetic acid.

9.3. Attaching sections to glass slides Routinely fixed paraffin-embedded sections are usually attached to slides by floating on a drop of a glycerine-albumin solution. Cryostat sections of unfixed tissues are picked up on unsubbed clean glass slides and frozen sections of fixed tissues on glycerine-albumin subbed slides. The immunohistochemical staining process is long and involves many incubation periods and much washing. Unless sections are very firmly attached to slides, they may lift off during the process. This may particularly occur during protease digestion or microwave heating. By far the most economical and best tissue adhesive that we have tested is 2% (v/v) aminopropyl-triethoxysilane in acetone. Using this method, however, we have still experienced some sporadic loss of sections or parts of sections from slides. We found that dipping the slides in concentrated hydrofluoric acid (HF) before silanisation considerably improves retention of sections.

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Glycerine-albumin. Dissolve 2.5 g of egg albumin, 0.25 g of NaCI in 50 ml of distilled water (warm to 37°C). Add 50 ml glycerol and 0.05 g thymol. The HF-APTES method of coating slides. In a fume hood, immerse frosted-ended slides, loaded into stainless steel racks, into concentrated HF for 15 s. Wash the slides thoroughly in tap water. Heat the slides at 125°C for 30 min in a convection oven to evaporate the thin layer of moisture always present on the surface. Immerse the slides in a 2% (v/v) APTES solution in acetone for 15 min. APTES-treated slides can be stored at RT after drying. Caution: HF is a very reactive acid. Always wear gloves and goggles and work in a fume hood. 9. 4. Preparing frozen tissue sections The procedure most frequenly used in this laboratory is the following: a small capsule is formed of metal foil and filled with OCT compound. The tissue is orientated as necessary near the surface of the viscous liquid and the capsule immersed in isopentane which has been cooled in liquid nitrogen. Allow to freeze completely. The tissue samples can be stored in air-tight containers at -70° C for up to one year and several weeks at -20°C. Fluctuations in temperature are damaging to stored samples and probably account for shorter storage at -20°C. Frequently for cryosectioning, morphology is clearly improved if the tissue is fixed and infused prior to sectioning. Fix small tissue samples in 4% PFA fixative for 2 h at 4°C. Following fixation, wash tissue samples twice in buffer. Place the samples in cold 30% (w/v) sucrose solution in buffer and store at 4°C. The tissue will initially float, but will sink if left overnight. After the tissue has sunk but not later than the day after fixation, embed the tissue in OCT and freeze it as described above. Prepare the sections of frozen tissues by standard techniques. The thickness of the sections will depend on the tissue being studied. In general, thinner sections are better for staining. Sections between 4 and 7/xm are commonly used. Sections are taken onto glass slides (uncoated clean glass

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slide for sections of unfixed tissues and glycerine-albumin or APTES-primed slides for frozen sections of fixed tissues). Air-dry sections for 15 min (cytoplasmic antigens) to 16 h (cell surface antigens). If slides are not to be stained immediately, they should be stored at - 2 0 ° C , preferably - 7 0 ° C . It is convenient to put slides back to back in pairs and then wrap them in aluminum foil before freezing. Before unwrapping, warm the slides up to room temperature in order to avoid the disastrous effects of water condensation. Then, if necessary, fix the slides in acetone or Chloroform at R T or 4°C for 10-20 min. Transfer to buffer without drying and immunostain. Alternatively, fix in 4% PFA for 10 min at 4°C. Wash 3 times, 5 min each, in buffer and immunostain. 9.5. Preparing paraffin tissue sections

Cut small blocks of tissue approximately 1 c m 2 x 0.4 cm. Fix in freshly prepared 4% paraformaldehyde, Bouin's fixative, 3 / 1 (v/v) ethanol-acetic acid, or PLP. Optimal fixation time is that which gives good morphology as well as a good signal-to-noise ratio after immunohistochemistry, and must be determined empirically for each of the samples. In general, tissues which are to be embedded in paraffin wax are fixed for 2 h to overnight, then embedded in wax on an automatic processor. 5 ~ m thick sections are affixed to slides by floating on a drop of a glycerine-albumin solution. If HF-APTES-treated slides are used, affix by floating on a drop of water. Antigens may be denatured by excessive or prolonged heating. For most work, drying for 8-24 h at 37°C is usually satisfactory. Particularly sensitive antigens may benefit from drying at RT. For any staining procedure, the complete removal of wax and of the dewaxing and rehydration agents is a vital step in the technique. If traces of waxes remain in the tissue, reagent penetration will be incomplete resulting in patchy staining across the section. Sections are dewaxed in three changes of xylene, each of 5 min duration. Three changes (5 min each) through graded ethanol (i.e 100%, 95%, 70%) will act as a good rehydradation sequence, before washing thoroughly in tap water.

9.6. Storage o f antibodies

For antibodies and antisera obtained commercially, the supplier's instructions should be followed. Most antibodies are stable for years when stored at -20°C. Working dilutions can be conveniently stored at 4°C where they are stable for a few weeks. 0.01% NaN 3 may be added to antibody solutions to prevent bacterial contamination. Antibodies should not be repeatedly thawed and refrozen and to avoid this the solutions should be divided into aliquots that are snap-frozen in liquid nitrogen before storing at 20 to 40°C. -

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9. 7. Staining procedures 9. 7.1. General considerations Slides should be treated with antibodies and conjugates in a damp chamber to minimize evaporation. It is imperative that the test area on the slide does not become dry at any stage because of the effects of high local salt concentration. For this reason when large batches of slides are processed, one slide at a time should be taken from the washing bath, surplus moisture removed and the next reagent applied before removing the next slide from the bath. All incubations of antibodies or detector molecules are performed at R T unless otherwise specified. If standardization is desired, the temperature should be carefully controlled. It is essential to determine the optimal dilutions of all the antibodies used. These will differ depending on a number of different factors such as the temperature of incubation, the length of time of incubation, the type of tissue section (frozen or fixed paraffin-embedded), the length of fixation, the type of fixation, etc. When using a new antibody, a number of sections should be stained using a set of dilutions which should be in geometric progression. The optimal dilution is that which stains a section close to the maximum strength with a minimal background. As a general rule, polyclonal antisera work well at 1 / 5 0 to 1/100 dilutions. After affinity purification, when the exact protein concentration is known, the working dilution should be 5-10 /xg/ml. Mono-

F. Plenatet al. /Journal of lmmunological Methods 174 (1994) 133-154 clonal antibodies are available in three main forms: hybridoma, culture supernatants, ascites and purified IgGs. The effective concentration of the antibodies in culture supernatant is usually very low. This antibody should be used either concentrated or at a dilution in the 1/5 to 1/10 range. Both ascites and purified monoclonal immunoglobulins are generally used very diluted (1 : 1000 to 1 : 10000). It is worth noting that higher antibody concentrations are usually used in immunohistochemistry than it is the case with other immunological methods. Because the antigen in tissue staining is fixed to a solid phase, the time needed for the antibody to fix the antigen will be longer than if both molecules are in solution. The incubation times can be adjusted for the experimental design, but seldomly will times less than 30 min yield efficient binding. Some workers prefer to incubate tissue sections with primary antibodies for 2-3 h at RT. Antibodies with a high level of activity for their specific antigens perform just as well at refrigerator temperature with extended incubation if diluted. This procedure is economical, increases the sensitivity and produces cleaner staining results. Most of the time, incubation times for detector molecules are around 30 min. Longer times may, however, be used for increased sensitivity and reduced cost. Most often, the sections are covered with 100150/xl of the antibody or detector molecule solutions. When working with expensive or precious reagents, incubation can be performed under coverslips using 5/xl of the antibody solution per cm 2 of coverslip. Washing is best accomplished in two or three steps in order to remove unreacted material efficiently. Unless this procedure is adopted, it is possible for very potent antibody preparations to produce false-positive tests by contamination in the washing solution.

9. 7.2. Blocking endogenous enzymatic activities If HRP- or AP-labeled detection methods are to be used, it may be necessary to block endogenous enzyme activities within the specimen before visualization of the immunoglobulin-linked enzymes. Complete inhibition of endogenous peroxidase activity can be achieved by treating the

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sections with a solution containing 0.3% (v/v) HzO 2 and 0.1% (w/v) NaN 3 in buffer for 10 min. Because the blocking procedures may harm some antigens, incubation with this blocking solution is preferably carried out after the incubation with the primary antibody. Indeed immunoglobulin determinants are relatively resistant to this procedure. When working with frozen sections it is often simpler to use the AP method if intense endogenous peroxidase from cells such as eosinophils exists in the material for examination, and if the peroxidase blocking interferes with sensitivity. To block endogenous AP activity 0.1 mM levamisole (or tetramisole) is included in the substrate solution. This reversible inhibitor does not diminish the activity of intestinal AP used to prepare the labeled antibody but does inhibit the activity of most other tissue phosphatases. If endogenous enzyme activity remains a problem after these blocking steps, the use of gold-labeled antibodies may be necessary.

9. 7.3. Reduction of free aldehydes Borohydride will unmask antigenic sites by the reduction of free aldehyde groups, thus reducing background staining due to non-specific binding of immunoreagents to these groups. Use 1% sodium borohydride in 0.1 M phosphate buffer pH 8.4 and treat sections for 30 min. It is important that the pH of the solution be greater than 8.2. Neutral or less alkaline solutions will not reduce all the aldehyde groups present in a section. Thoroughly wash in buffer. This pretreatment will also reduce the autofluorescence due to fixation in glutaraldehyde. Free aldehyde groups can be also quenched by incubating the sections in a 1% (w/v) solution of glycine in TBS pH 7.4; the aldehyde groups reacting with the free amino groups of glycine. 9. 7.4. Exposure of hidden antigens Sometimes treatment of a section of fixed tissue with a proteolytic enzyme will 'reveal' a previously detected antigen. The use of a proteolytic enzyme adds an extra-variable to the procedure which is difficult to control precisely. As a general practice we would not recommend it. However, in diagnostic pathology, the only available

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tissue may already have been fixed in formalin and embedded in paraffin wax and these procedures may not be optimal conditions for a particular antigen. In these circumstances, there is little choice but to try this approach. A suitable recipe is as follows: a trypsin solution is prepared by dissolving 0.1% ( w / v ) trypsin and 0.1% ( w / v ) CaC12 in TBS p H 7.8. Preheat the solution in a Coplin jar to 37°C in a water bath. Place the rehydrated sections in the glass slide jar and incubate for a time that should be determined for each tissue by trial and error. Overdigestion with proteolytic enzymes damages tissue morphology. In our experience the trypsin sold by Prolabo works well but less pure preparations of trypsin are known to be less efficient. For recovering masked antigens by microwave heating, the rehydrated sections are placed in a plastic jar filled with 10 mM citrate buffer p H 6.0. The slides are processed 3 - 5 times for 5 min each at 750 W. Slides should not dry during the incubation in the microwave oven. If necessary, stop incubation and add distilled water to replace evaporated quantity. Sections are allowed to cool in the tray at R T for about 20 min. They are then rinsed in TBS.

9.7.5. Nonfat dried milk solution for reducing background staining The mixture is: 30% ( w / v ) nonfat dried milk, 3% ( w / v ) BSA, 0.3% (v/v) Tween 20, 0.1% (w/v) NaN 3. An even better signal-to-noise ratio can be obtained by the addition of 3.5 mg or 500 units of sodium h e p a r i n a t e / m l of blocking solution. Caution: N a N 3 is poisonous. It shoud be handled with great care wearing gloves, and solutions containing it should be clearly marked. 9.7.6. lndirect two-step immunofluorescent techniques Proceed as follows: (1) bring the attached sections to the buffer wash stage; (2) wipe the slides dry except for the area of section; place the slides on a rack in a humid chamber; (2) block nonspecific binding if required; for example, apply to the sections a drop of nonfat dried milk or of normal serum from the species supplying the second antibody diluted 1 / 3 0 in buffer; (3) re-

Fig. 1. Lung epidermoid carcinoma. Cellular membranes of macrophages (arrows) that are scatterred within the stroma were labeled with a mouse monoclonalantibody to MAX-3 (a generous gift from M. Kreutz, Department of Oncology and Hematology,Heidelberg). Paraformaldehyde-fixedfrozen section. Indirect three-step immunoenzymatic technique with HRP-labeled secondary and tertiary antibody. Color development with the DAB-H202 method. Hematoxylin counterstaining. Magnification: × 400. move excess milk or protein and do not wash; (4) apply unlabeled primary antibody, optimally diluted, for 1 h or overnight at R T or 4°C; (5) rinse in 3 baths of buffer, 5 min each; (6) apply a drop of a fluorescent-labeled second antibody that will combine with immunoglobulins of the species in which the primary antibody was raised at a predetermined dilution and incubate in the dark for 30 min to 1 h; (7) rinse in 3 baths of buffer, 5 min each; (8) mount in an anti-fade mounting medium.

9. 7. 7. Indirect three-step immunoenzymatic or immunofluorescent techniques (Fig. 1) Proceed as follows: (1) bring the attached sections to the buffer wash stage; (2) block nonspecific binding if required; for example, apply to sections a drop of nonfat dried milk or of normal serum from the species supplying second antibody diluted 1 / 3 in buffer; (3) remove excess protein solution and do not wash; (4) apply unlabeled primary antibody, optimally diluted, for 1 h or overnight at R T or 4°C; (5) when working with peroxidase-labeled antibody, block endogenous peroxidase by dipping the slides in a solution containing 0.3% (v/v) H 2 0 ~ and 0.1% (w/v) NaN 3 in buffer for 10 min; rinse in 3 baths of

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buffer; (6) apply a drop of a conjugated second antibody that will combine with immunoglobulins of the species in which the primary antibody was raised, at a predetermined dilution for 0.5-1 h; (7) rinse in 3 baths of buffer, 5 min each; (8) apply a drop of a conjugated tertiary antibody that will combine with immunoglobulins of the species in which the secondary antibody was raised at a predetermined dilution for 30 min to 1 h; (9) wash in buffer, 3 changes, 5 min each; (10) when working with fluorochrome-labeled antibodies mount in an anti-fade mounting medium; (11) when working with enzyme-labeled antibodies, carry out the visualization procedure, counterstain if necessary and mount.

9.7.8. Staining method for PAP or APAAP procedures Proceed as follows: (1) bring the attached section to the buffer wash stage; (2) block nonspecific binding if required, e.g., apply to section a drop of normal serum from species supplying second antibody diluted 1/3 in buffer; (3) discard the excess protein solution and do not wash. Wipe slide dry except for the area of section; place the slides on a rack in humid chamber; (4) apply unlabeled primary antibody, optimally diluted, for 1 h or overnight at RT or 4°C; (5) rinse in 3 baths of buffer, 5 min each; (6) when working with PAP complexes, block endogenous peroxidase by dipping the slides in a solution containing 0.3% (v/v) H 2 0 2 and 0.1% (w/v) NaN 3 in buffer for 10 min; rinse in 3 baths of buffer; (7) apply a drop of an unlabeled second antibody that will combine with immunoglobulins of the species in which the primary antibody was raised at a predetermined dilution for 30 min to 1 h; (8) rinse in 3 baths of buffer, 5 min each; (9) apply a drop of optimally diluted PAP or APAAP complexes prepared with antibodies of the same species and isotype as those of the primary antibody, at RT for 30 min; (10) wash in buffer, 3 times, 5 min each; (11) carry out the visualization procedure; counterstain if necessary and mount. 9. 7.9. Detection of antigens by the streptavidin-biotin techniques Proceed as follows: (1) remove wax and bring the attached section to the buffer wash stage; (2)

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(optional) block non-specific binding sites, e.g., apply to sections a drop of nonfat dried milk solution for 30 rain; (3) discard the excess milk solution and do not wash; (4) apply the primary antibody, optimaly diluted, at RT or 4°C for 1 h to overnight; (5) rinse in 3 baths of buffer, 5 min each; (6) when working with peroxidase-labeled antibody, block endogenous peroxidase by dipping the slides in a solution containing 0.3% (v/v) H202 and 0.1% (w/v) NaN 3 in PBS for 10 min; (7) rinse in 3 baths of buffer, 5 min each; (8) apply biotinylated second antibody, optimally diluted for 30 min; (9) rinse in 3 baths of buffer, 5 min each; (10) apply HRP-conjugated streptavidin or AP-conjugated streptavidin, optimally diluted for 30 rain; (11) rinse in 3 baths of buffer, 5 min each; (12) apply the right substrate solution to give a colored end-product; (13) wash in tap water, counterstain and mount.

9.7.10. Indirect immunogold method with silver enhancement for light microscopy of conventionally fired paraffin sections Springall et al. (1984): (1) dewax sections and immerse in water; (2) treat with Lugol's iodine (1% iodine in 2% potassium iodide), 5 rain; this oxidation step is essential when working with formalin-fixed paraffin sections; (3) rinse in tap water; (4) treat with 5% aqueous sodium thiosulfate until the sections become colorless; (5) rinse in tap water; (6) wash in TBS containing 0.5% Triton X-100, 3 changes of 5 min each; (7) incubate in normal serum from species providing second antibody, diluted 1/3, 10 min; (8) drain off serum and wipe slide dry except for the area of section. Place the slides on a rack in a humid chamber and incubate in appropriately diluted primary antibody at 4°C or RT for 1-24 h; (9) wash in TBS, 3 changes of 5 min each; (10) apply a drop of a second antibody that will combine with immunoglobulins of the species in which the primary antiserum was raised at a predetermined dilution for 30 min to 1 h; (11) wash in TBS, 3 changes of 5 min each; (12) apply a drop of a tertiary antibody that will combine with immunoglobulins of the species in which the secondary antibody was raised; this tertiary antibody, adsorbed to 1 nm diameter colloidal gold parti-

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cles, is appropriately diluted in 0.01 M Tris-HCl buffer pH 8.2, containing 0.8% BSA; incubate for 1 h; (13) wash in 0.01 M Tris-HCl buffer pH 8.2, 3 changes, 5 min each, and then in distilled water, 6 changes of 5 min each; (14) carry out silver reaction as described below. 9. 7.11. Immunohistochemical detection o f B r D U or IUdR incorporated into D N A Proceed as follows: (1) fixed tissue samples which have been exposed in vivo to IUdR in ethanol/acetic acid (3/1, v / v ) for 12 h at RT; (2) embed in paraffin following regular procedures; (3) dewax, rehydrate and bring to distilled water; (4) denature DNA by immersing the slides in 4 N HC1 for 15 min at RT; (5) carefully wash in 3 baths of tap water, 5 min each; (6) incubate the slides in nonfat dried milk solution for 30 min at RT; (7) discard the excess milk solution and do not wash; (8) apply a monoclonal antibody to BrDU optimally diluted, at RT for 1 h; (9) wash in buffer, 3 times, 5 min each; (10) reveal tissuefixed primary antibody either with an indirect three-step immunoenzymatic technique or the streptavidin-biotin method. Nota bene: in humans, IUdR (2.4 g diluted in 240 ml 5% dextrose in water (pH 9.5) is intravenously infused over a 24 h period; mice are intraperiteonally injected, 24 h before removing the sample to be studied, with 40 /xg I U d R / g body weight, in distilled water pH 9.5. 9. 7.12. Developing enzymatic activities 9. 7.12.1. Peroxidase substrates. (a) Diaminobenzidine tetrahydrochloride. This electron donor is the most commonly used substrate of peroxidase and one of the most sensitive. It gives a brown precipitate which is insoluble in water, ethanol and toluene. DAB staining is compatible with a wide range of common histological stains. Dissolve 6 mg of DAB in 10 ml of a 0.05 M Tris-HCl buffer or 0.1 M PBS (pH 7.6). The choice of phosphate or Tris buffer is not critical. The solution should be very pale, almost white. However, darkly colored solutions do not always give non-specific background staining. Add 3 /xl of 30% H 2 0 z. Avoid contact of the strong

H 2 0 2 with skin or clothing. H 2 0 2 is unstable, and old stock should not be used. Decomposition is accelerated by chemical contamination, especially by contact with metal. It is important to use the recommended concentration of H 2 0 2 in the medium in order to prevent the formation of colored products due to catalase. Cover the sections and incubate in the dark for 5-10 min at RT. It is possible to microscopically observe and control the product formation but this is not recommanded in view of the possible DAB carcinogenicity. Wash in tap water or PBS. Counterstain if necessary. Mount in Eukitt. Control sections should be incubated with DAB in the absence of H 2 0 2. On unfixed sections a positive reaction in the absence of H 2 0 2 can be due to cytochrome oxidase. DAB is always handled with caution, although it is probably not carcinogenic. It may be purchased in rubber-capped vials, each containing a pre-weighed amount or as tablets. Any spillage may be neutralized (oxidized) with 5% sodium hypochlorite (household bleach). (b) 3-Amino-9-ethylcarbazole. This gives a red precipitate soluble in ethanol and toluene. Stock solution (stable at RT) is: 0.4% (w/v) 3-amino-9ethylcarbazole in dimethylformamide. Dissolve 0.5 ml of stock solution in 9.5 ml of 0.05 M acetate buffer pH 5.0. Just before use add 10/xl of 30% H 2 0 2. Filter on the sections and incubate at RT for 5-10 min. Counterstain lightly in Mayer's hemalun. Do not differentiate the hemalun in acid-alcohol. Mount in an aqueous medium. (c) 4-Chloro-naphthol. This can be used if a blue color is required. 3 mg of the 4-chloro-naphthol in 0.1 ml of absolute ethanol are added while stirring to 10 ml of 0.05 M Tris-HC1, pH 7.6. Before use, add 5 /zl of H 2 0 2 30%. A white precipitate forms. Filter out the white precipitate before use. Incubate the sections at RT for 30 min. The precipitate is soluble in alcohol and xylene. Mount the section using an aqueous mounting medium. 9.7.12.2. Alkaline phosphatase substrates. (a) Naphthol-AS-BI-phosphate/fast red TR salt (or fast blue RR salt). Naphthol-AS-BI-phosphate/ fast red or fast blue substrates produce an intense

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red (or blue) product that is soluble in alcohols as well as organic solvents. Dissolve 2 mg naphtholAS-MX phosphate in 0.2 ml of dimethylformamide in a glass tube. Add 9.8 ml of 0.1 M Tris-HCl buffer, pH 8.2. Fresh solution is prepared each time. Immediately before staining, dissolve Fast red TR salt (or Fast blue RR salt) at a concentration of 1 m g / m l and filter directly onto the slides. If it is required to block endogenous alkaline phosphatase activity, levamisole should be added to the substrate solution at a final concentration of 1 mM. (b) Naphthol-AS-MX-phosphate/New Fuchsin. This method produces an intense red product that is insoluble in alcohols as well as organic solvents and is compatible with a range of histochemical stains. Add the following to a tube at 4°C: 0.5 ml of a 5% solution of New Fuchsin in 2 N HC1 and 1.25 ml of a freshly prepared 4% solution of sodium nitrite. After 60 s this solution is added to 250 ml of 0.05 M Tris-HC1 (pH 8.7) containing 90 mg of levamisole. Then add to the mix 125 mg of naphthol-AS-MX phosphate, freshly dissolved in 1.5 ml dimethylformamide. This solution is then filtered and used immediately for staining. Incubation with substrate is continued up to 30 rain with continual agitation. (c) Bromochloroindolyl phosphate/nitroblue tetrazolium. The B C I P / N B T substrate generates an intense black-purple precipitate at the site of enzyme bonding. The reaction proceeds at a steady rate, thus permitting accurate control of the development of the reaction. This also permits the relative sensitivity to be controlled by the length of incubation. Prior to developing the tissue staining, prepare the three stock solutions: (1) NBT: dissolve 50 mg of NBT in 1 ml of 70% dimethylformamide. (2) BCIP: dissolve 75 mg of BCIP in 1 ml of 100% dimethylformamide. (3) AP buffer: 100 mM NaCI, 5 mM MgCI2, 100 mM Tris-HCl (pH 9.5). The stock solutions of BCIP and NBT are not very stable, even when kept at -20°C. It is good practice to prepare fresh solutions every month. Just prior to developing, prepare fresh substrate solution: add 99 /xl of NBT stock to 22.5 ml of AP buffer. MIX well and add 75/xl of BCIP stock. Incubate the tissue sections in Coplin jars. Develop at RT in the dark with

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agitation until the stain is suitably dark. Periodic monitoring under the microscope may be necessary for some antigens. A typical incubation would be 30 min. To stop the reaction, rince with TBS containing 20 mM EDTA which chelates the Mg 2+ ions, or in tap water if another antigen does not have to be detected. Mount in a watersoluble mountant.

9. 7.13. Detecting gold-labeled reagent (1) Rinse the specimen in distilled water briefly. (2) For silver enhancement, transfer the sample to a darkroom equipped with a safe light. To prepare the developer, mix 20 ml of 1 M citrate buffer (1 M citric acid, 0.5 M trisodium citrate), pH 3.5; 33 ml of 30% (w/v) gum acacia, 15 ml (w/v) of silver lactate (0.11 g in 15 ml); 15 ml (w/v) of hydroquinone (0.85 g in 15 ml); 15 ml of distilled water. All solutions are prepared in distilled water and mixed in the above order. The solutions of silver lactate and hydroquinone should be prepared immediately before use. (3) Immerse the slides in silver solution and develop for up to 30 min whilst observing by microscopy. (4) Rinse in 2.5% sodium thiosulfate. (5) Wash throroughly in distilled water for 10 min. (6) Counterstain with methyl green, dehydrate, clear and mount. Nota bene: Recently several commercial companies have introduced silver enhancement kits that do not require the use of a darkroom. 9. 7.14. Counterstaining It is general practice to counterstain the nuclei in order to reveal the architecture of the tissue. If the immunohistochemical stain is red or brown, either hematoxylin or Mayer's hemalun is used. If the immunostain is blue, then chloroform-extracted methyl green is preferred (see standard books of histochemistry for recipe). The development of fluorescent nuclear counterstains makes it possible to visualize unstained areas after immunofluorescent procedures. Ethidium bromide and propidium iodide intercalate with the nuclear DNA to form a highly fluorescent compound which has a bright orange fluorescence when examined with filter systems employed with fluorescein conjugates. The staining is accomplished

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by merely immersing the slides immediately before mounting in a 1 / z g / m l solution of the halide in buffer. Since the fluorescence excitation and emission of the halides is indistinguishable from that of T R I T C by microscopy, it is necessary to use an alternative nuclear counterstain with this fluorochrom. Bisbenzidine (Hoechst 33258 or 33542), used at a concentration of 20 / x g / m l or less is satisfactory and produces blue nuclear fluorescence when excited with UV light.

9. 7.15. Mounting the coverslips If the reaction product is insoluble in ethanol and xylene, the slides are brought to water through ethanol and xylene. The coverslips are mounted in a natural or synthetic resin such as Eukitt. If not, they are left in water and the coverslips mounted in glycerine jelly or a similar water-based mountant. To make glycerine jelly: dissolve 10 g of gelatine in 60 ml distilled water using gentle heat; add 70 ml of glycerol and 0.25 g of phenol and mix well; aliquot in 10 ml batches and store at - 2 0 ° C . Before use, melt in a water bath. Avoid shaking as this creates air bubbles. When working with fluorescent reagents add p-phenylenediamine at a concentration of 10 -2 M in glycerine jelly. Alternatively, use the following polyvinyl alcohol anti-bleaching solution (Gorsky and Borosy, 1989): dissolve 2.4 g polyvinyl alcohol ( M r 30-70000) in 12 ml 0.2 M% Tris-HCl p H 8.5 containing 6 ml methanol and 6 g dimethyl sulfoxide. Enclose the mixture in a tube, heated in a boiling water bath for 10 min. Store at -20°C. On the day of use, add p-phenylenediamine to an aliquot at 2 m g / m l . 10. Comments

It is difficult to make generalizations, concerning the choice of the best method to be used to detect a given antigen in human or experimental samples. When using a new antibody, the first step is to determine the optimum conditions for fixation and processing. These conditions being defined, the detection system has to be chosen, knowing that it is mainly a historical rather than a practical fact that immunofluorescence is used on

cryostat sections and immunoenzymatic or immunogold techniques on paraffin sections. All these procedures can be utilized on material prepared by either technique. It must be emphasized that the use of epi-illumination and narrow band filter systems plays an essential role in eliminating background fluorescence with such tissues. The resolution and sensitivity of cell staining depend on the methods used for the antibody detection. High spatial resolution can be obtained using fluorochrome-labeled antibodies, where images of subcellular structures can be studied at magnifications beyond the limit of resolution of the transmitted light microscope. Because the signal generated by immunoenzymatic techniques is detected by differential absorption rather than emission of light and because the insoluble product of the chromogenic substrate will be deposited on a small area around the site of enzyme localization, this detection method can never approach the resolution of fluorescent techniques. The sensitivity of the immunofluorescent staining methods is not inherently any lower than that of the immunoenzymatic or immunogold sandwiches of similar geometry. Fluorochromes are not less efficient as labels than enzymes. Still higher sensitivity can be obtained by using a confocal microscope whose laser sources can be focussed pointwise. The most commonly cited drawback of fluorescent procedures (i.e., that they yield preparations which are impermanent) is for some reason greatly exaggerated when mounted with an antibleaching solution. Fluorescently labeled sections retain their label for months if properly stored at -20°C. Enzyme or gold-labeled antibodies provide sensitive antigen detection and require only a suitable substrate and a light microscope for their detection. The advantages of these methods are that sections have an indefinite (or at least extended) shelf life, and morphology and labeling may be examined using the same type of illumination. Immunogold-silver staining is superior to the procedures using enzyme-labeled antibodies or enzyme-anti-enzyme complexes in sensitivity but is often difficult to control. Only minor differences in sensitivity and efficiency exist between the triple-step indirect, streptavidin/biotin and the unlabeled PAP or APAAP methods. There is

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no right or wrong choice of enzymatic label. Selection should be guided by the tissue to be studied. AP would probably be a poor choice if a study is to be m a d e of the gut since there is a high concentration of this enzyme in the tissue. H R P is usually best avoided if the tissue contains large amounts of endogenous peroxidase or brown pigment. However, it is our experience that methods using AP as label are more sensitive than equivalent techniques with H R P . They are also more expensive.

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(7) Microscopes: (a) Leica Instruments, Postfach 1120, Heidelberger Strasse 17-19, D-6907 Nussloch, Germany. Tel.: (0 62024) 143-0; Fax: (0 62 24) 1 00 15. (b) Zeiss instruments, Postfach 1369 and 1380, D-7082 Oberkochen, Germany. Tel. 49 073 64 20 70. (8) Cryostat: 2040 Cryocut, Leica Instruments, Postfach 1120, Heidelberger Strasse 17-19, D-6907 Nussloch, Germany. Tel.: (0 62024) 143-0; Fax: (0 62 24) 1 00 15.

Acknowledgement 11. Reagents and equipment Antibodies and conjugated detector molecules: a listing of commercially available antibodies and conjugated detector molecules can be found in Linscott's catalogue of immunological and biological reagents, 4877 Grange Road, Santa Rosa, C A 95404, USA. Tel.: 707 544 9555. Most of the chemicals used in the techniques reported above may be obtained from Sigma Chemical Company, P.O. Box 14509, St. Louis, M O 63178, USA. Tel.: 314 771 5750; Fax: 31477. However, other sources are as follows. (1) Trypsin, picric acid, paraformaldehyde from Prolabo, 65 Bd Richard le Noir, 75011 Paris, France. Tel.: (1) 48 07 38 21; Telex: Prolabo 250584F. (2) Entellan and dimethyl sulfoxide from Merks, Frankfurter Strasse 250, D-6100 Darmstadt 1, Germany. Tel.: (0) 6151-72-0. (3) Eukitt from Labonord, Z I du Fort, 21 rue du H a u t de la Cruppe, BP 648-59656, Villeneuve d'Ascq Cedex, France. Tel.: 20 56 02 02. (4) Acacia powder (gum arabic) from Aldrich Chemical Company, P.O. Box 2060, Milwaukee, WI 53201, USA. Tel.: 800-558-9160; Fax: 414-273-4979. (5) O C T compound from Miles Diagnostic Division, Elkhart, IN 46515, USA. Tel. (1) 219264-81-11. (6) Silver e n h a n c e m e n t kits from A m e r s h a m International, R e s e a r c h Products Division, White Lion Road, A m e r s h a m , Buckinghamshire HP9LL, UK. Tel.: (44) 50-240444444; Fax: (44) (0)296-85190.

This work was supported by a grant from the Ligue Lorraine de Lutte contre le Cancer.

References Andreesen, R., Klaus, J., Osterhold, J. and Emmrich, F. (1986) Human macrophages maturation and heterogeneity: analysis with a newly generated set of monoclonal antibodies to differentiation antigens. Blood, 67(5), 12571264. Barclay, A.N., Beyers, A.D., Birkeland, M.L., Brown, M.H., Davis, S.J., Somoza, C. and Williams, A.F. (1992) The Leucocyte Antigen Factsbook. University of Oxford, Oxford, 448 pp. Bullock, G.R. and Petrusz, P. (1983a) Techniques in Immunocytochemistry, Vol. 2. Academic Press, London. Bullock, G.R. and Petrusz, P. (1983b) Techniques in Immunocytochemistry, Vol. 3. Academic Press, London. Cordell, J.L., Falini, B., Erber, W., Gatter, K.C. and Mason, D.Y. (1984) Immunoenzymatic labeling of monoclonal antibodies using complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP) complexes. J. Histochem. Cytochem. 32, 219-229. De Jong, A.S.H., Van Kessel-Van Mark, M. and Raap, A.K. (1985) Sensitivity of various visualization methods for peroxidase and alkaline phosphatase activity. Histochem. J. 17, 119-130. De Mey, J. (1983) Colloidal gold probes in immunohistochemistry. In: J.M. Polak and S. Van Noorden (Eds.), Immunocytochemistry. Practical Applications in Pathology and Biology. Wright PSG, Bristol, pp. 82-112. Duprez, A., Borrelly, J., Grosdidier, G., Hofmann, M. and Boileau, S. (1989) Etude in vivo de la prolif6ration cellulaire de tumeurs humaines, par histo-immunod&ection de la 5-d6soxyuridine incorpor6e dans I'ADN. CR Acad. Sci. 308 (Ser. III), 313-319. Harlow, E.D. and Lane, D. (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

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Gorbsky, G.J. and Borisy, G.G. (1989) Hapten-mediated immunocytochemistry: the use of fluorescent and nonfluorescent haptens for the study of cytoskeletal dynamics in living cells. Methods Cell Biol. 29, 175-193. Matthews, J.B. and Mason, G.I. (1984) Influence of decalcifying agents on immunoreactivity of formalin-fixed, paraffin-embedded tissue. Histochem. J. 16, 771-787. Polak, J. and Van Noorden, S. (1983) Immunocytochemistry. Practical Applications in Pathology and Biology. Wright PSG, Bristol. Shi, S.R., Key, M.E. and Kalra, K.L. (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhance-

ment method for immunohistochemical staining based on microwave oven heating of tissue sections. J. Histochem, Cytochem. 39, 741-748. Springall, D.R., Hacker, G.W., Grimelius, L. and Polak, J. (1984) The potential of the immunogold-silver staining method for paraffin sections. Histochemistry 81, 603-608. Sternberg, L.A., Hardy, P.H., Cuculis, J.J. and Meyer, H.G. (1970) The unlabeled antibody enzyme method for immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in the identification of spirochetes. J. Histochem. Cytochem. 18, 315-333.