Quantitation of immunogold labeling

Micron and Microscopica Acta, Vol. 23, No. 1/2, pp. 1-16, 1992. Printed in Great Britain.

0739-6260/92 $5.00+0.00 © 1992 Pergamon Press Ltd

Q U A N T I T A T I O N OF I M M U N O G O L D LABELING M. A. HAYAT Department of Biology, Kean College of New Jersey, Union, NJ 07083, U.S.A. (Received 25 June 1991; revised 12 August 1991)

Abstract--Most of the published, quantitative immunogold labelingstudies do not reflectthe true concentration of antigen in a given cell compartment. Presently, the majority of the data on labelingdensity providesa potential way to followthe distribution of antigens, but does not indicate the quantitative concentration of the antigen present. The majority of the published quantitative data is semiquantitative. Ideal quantitative data must relate the intensity of the immunoreaction to the concentration of the underlying antigen population. Since this requirement is not fulfilled by most of the available methods, caution is warranted in interpreting the immunogold quantitative data. The difficulty in obtaining reproducible quantitative data is also due to the fact that preparatory procedures are quantitatively unknown parameters with respect to quantitative immunogold postembedding and preembedding labeling.

INTRODUCTION In 1971, Faulk and Taylor inaugurated the use of colloidal gold as an immunocytochemical marker for transmission electron microscopy. Since that time, the use of this marker for immunoelectron microscopy has advanced by a quantum leap. In fact, colloidal gold has become the most important and the most widely employed methodology for the in situ localization of cellular and viral macromolecules. Colloidal gold is a truly universal marker since it is applicable to bright field (Scopsi, 1989; Hacker, 1989) and conventional transmission electron microscopy (Faulk and Taylor, 197 l) as well as to high voltage electron microscopy (Takata and Hirano, 1989; G o o d m a n et al., 1991). Its applications are extended to dark-field, epipolarization (De Mey, 1983; De Waele, 1989) and fluorescent microscopy (Horisberger and Vonlanthen, 1979). Reflection contrast microscopy has also been used for visualizing silverenhanced colloidal gold particles (Hoefsmit et al., 1986; Velde and Prins, 1990). Colloidal gold is also employed as a marker for scanning electron microscopy in the secondary (Horisberger, 1989) and backscattered modes (de Harven and Soligo, 1989; N a m o r k , 1991). Another approach is the use of an energy-dispersive analyzer for localizing and identifying colloidal gold particles (Eskelinen and Peura, 1991). Using colloidal gold, correlative light and electron microscopy can be carried out on the same section or on an adjacent section cut from the same specimen block (Mar and Wight, 1989). Correlative video-enhanced light microscopy, high voltage transmission electron microscopy, and low-voltage high resolution scanning electron microscopy have been used for studying the binding of colloidal gold-labeled fibrinogen to platelet surfaces (Simmons et al., 1990). The methodology of colloidal gold labeling is applicable to a wide variety of ligands such as proteins including enzymes, lipoproteins, immunoglobulins, lectins, and hormones. The application of protein A-gold to label antibodies bound to surface

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antigens is well known (Romano and Romano, 1977). Equally well known is the use of the immunoglobulin-gold complex (Faulk and Taylor, 1971). The preparation of immunoglobulin~old is more complex than that of protein A-gold, since polyvalent immunoglobulins consist of a mixture of molecules with a wide range of isoelectric points. Immunogold labeling is also useful for studying viruses and viral antigens (Stannard et al., 1982; Kjeldsberg, 1989). The immunonegative staining procedure in conjunction with colloidal gold labeling can be employed for the immunological study of small specimens such as bacteria and viruses (Mfiller and Baigent, 1980; Beesley, 1989; Kjeldsberg, 1989). Colloidal gold is the marker of choice in label-fracture cytochemistry (Kan and Pinto da Silva, 1989) and for studies using thin cryosections (van Bergen en Henegouwen, 1989; Pietschmann et al., 1989; van den Pol et al., 1989). This marker can additionally be used in conjunction with carbon evaporation and rotary platinum shadowing after immunolabeling (van den Pol et al., 1989). Radioactive colloidal gold has been developed as a tracer for estimating cellular pinocytosis (Roberts et al., 1977). A number of probes displaying highly specific binding properties can be used in combination with colloidal gold. Lectins are highly specific probes for carbohydrates, but these probes are not electron dense. This limitation is circumvented by using lectin gold complexes. Such complexes have been employed for cell surface studies (Horisberger, 1981) and for the detection of intracellular receptors (Roth, 1983; Benhamou, 1989). Lectins can be used either directly bound to colloidal gold or together with appropriate glycoprotein-gold complexes. Another example is the use of gold-substituted silver peroxidase intensification (van den Pol et al., 1989). Peroxidase immunostaining by itself results in a thin layer of precipitated reaction product, which does not allow clear visualization of antigenic determinants. Another approach involves the use of a streptavidin-gold reagent for the detection of biotinylated lectins and second antibodies (Pettitt and Humphris, 1991). This system has three advantages: (1) the signal amplification inherent in the streptavidin-biotin carrier interaction provides a reliable detection of a target moelcule even when it is present at a quantitatively low concentration, (2) only a simple detector reagent with gold particles of the desired size is required, and (3) since streptavidin can be efficiently linked to fluorescent and chromogen detectors as well as to colloidal gold, multiplelabeling studies can be carried out. Troxler et al. (1990) have compared the sensitivity of biotinylated DNA and RNA probes, detected with streptavidin or anti-biotin antibodies and using colloidal gold as the label. Colloidal gold can be used either coupled directly to the biotin-detecting substance (streptavidin or antibody) or to an anti-species antibody. In this approach no further amplification (e.g. bridging antibodies) of the signal is required. In situ hybridization can be used in conjunction with streptavidin-gold as an electron dense marker to localize both DNA and RNA (Binder et al., 1986). Wolber and Beals (1989) have indicated achieving quantitation with streptavidin-gold labeling for ultrastructural in situ nucleic acid hybridization. Enzymes tagged with gold particles constitute a specific probe for the cytochemical detection of corresponding substrate molecules (Bendayan, 1984, 1989). The enzyme-gold approach is complementary to autoradiography (Bendayan and Puvion, 1983). Two types of immunogold labeling are available. Postembedding labeling is favored for detecting cytoplasmic and extracelluar antigens, while preembedding labeling is preferred for localizing cell surface-associated antigens. Preembedding labeling of cells in suspension usually results in increased labeling efficiency of surface antigens (Ferrari et al., 1989) compared with that obtained by using postembedding labeling of thin resin or cryosections. Although quantitation has been carried out using both of these methods, the following discussion pertains primarily to postembedding labeling. The estimation of quantitation of antigens with immunogold labeling is discussed below.

Quantitation of immunogold labeling

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QUANTITATION One of the most important aspects of colloidal gold methodology is the possibility of quantifying the concentration of antigens. This is possible because gold particles linked to antibodies provide high spatial resolution for quantitation. Theoretically, it is expected that the labeling density of cell structure X containing n molecules of the antigen/lxm 3 will be one-half of that of cell structure Y containing 2n concentration of the antigen (Slot et al., 1989a). However, this is true only when the conditions of processing of cell structures X and Y are identical and antigen concentrations are lower than those causing significant steric hindrance among immunomarkers. It is important to know the antigen threshold. In practice, it is difficult to control various factors that influence the relationship between antigen concentration and labeling density. These factors are discussed later. Presently, quantitation in most studies does not mean that the number of gold particles is the same as the number of antigens on the specimen surface. The efficiency of labeling as yet has not approached the 'one antigen, one gold' (Howell et al., 1987) level even for a plasma membrane antigen, and protein A-gold complexes of any size have not attained saturation of the available binding sites. The question is how many antigens or epitopes on an average in the entire depth of a thin section become labeled by gold particles? Does a stoichiometric relation exist between the concentration of the antigens present and the labeling achieved? Presently, we are not sure. The quantitation of protein-gold complexes and correct analysis of their affinity and numerical relationship to cellular binding sites remain as yet unsolved problems. However, a number of methods have been developed for obtaining quantitation of immunogold labeling with various degrees of success, which are summarized later. The ultimate goal of quantitative immunocytochemistry is to accomplish absolute quantitation of antigen molecules or epitopes. In practice, quantitation implies a linear relationship between the number of bound gold markers and the number of antigenic molecules on the surface of the specimen section. To achieve reasonable quantitation, the reproducibility and the highest labeling (staining) efficiency are prerequisites. The efficiency of labeling is indicated by the percentage of antigen molecules in a section marked with gold particles. To obtain quantitative data from thin cryosections it is essential that either the efficiency of labeling is known or it is equal in each compartment of the cell. In the latter case true quantitative data are attainable using a proper calibration of the system, which should be included in each experiment to annul differences in exogenous factors. Such an approach is useful when a high labeling efficiency is obtained, but this is often incompatible with the necessity of adequate fixation to retain proteins of interest in the specimen. Furthermore, a demand for high labeling efficiency is only valid when the measured labeling densities are not statistically significant and are different from background levels. Since immunogold particles are uniformly restricted to the section surface irrespective of the type of embedding resin used (Kellenberger and Hayat, 1991), the quantitation data refer to the density of the bound gold particles per area of a cell compartment or an organelle only at the section surface. However, according to Posthuma et al. (1987), IgG molecules may penetrate into resin sections, thus yielding high labeling efficiencies. This advantage, if true, seems to be valid only for watermiscible resins such as Lowicryl K4M and LR White (G. Posthuma, unpublished observations). Nevertheless, overwhelming published evidence does not support these observations. In the case of thin cryosections, although the antibody penetration is basically limited to their surface, such penetration somewhat varies in different cell compartments. Immunogold particles can penetrate the outer surface of the plasma membrane in thin cryosections using pellets of unembedded cells (Griffiths et al., 1990).

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Factors affecting quantitation The maximum degree of labeling is necessary to accomplish quantitation since it is difficult to distinguish between lack of antigens and imcomplete labeling. A wide variety of factors control the relationship between antigen concentration and labeling density, for labeling efficiency is dependent upon a large number of factors. Both exogenous and endogenous factors determine the efficiency of the immunoreaction. Exogenous factors usually influence labeling efficiency equally throughout the specimen under optimal conditions including homogeneous fixation and complete penetration as well as polymerization of the embedding medium (Slot et al., 1989a). Endogenous factors, on the other hand, can affect immunolabeling of various cells and cell compartments to different degrees. Consequently, variations in labeling efficiencies among cellular compartments are not uncommon. Thus, endogenous factors may obscure the relationship between labeling density and antigen concentration when different cellular compartments are involved (Slot et al., 1989a). Exogenous factors. Exogenous factors are derived primarily from preparatory procedures such as chemical fixation or cryofixation, dehydration, embedding, characteristics of the immunoreagents, specificity and concentration of the antibody, size of the gold particles, and the protocols of incubation. Variations in specimen processing is a major obstacle in obtaining reproducible quantitative data, since optimization of labeling parameters for an antigen is a prerequisite for quantitation. Experiments have been carried out for optimizing these parameters by using polymerized BSA as an artificial substrate into which a desired antigen is incorporated by a simple soaking procedure (Valnes et al., 1984; Gagne and Miller, 1987). Such BSA blocks can be treated identically to pieces of tissue. This approach allows quantitative determination of optimal processing conditions in conjunction with colloidal gold labeling. Since test can be performed with BSA in an identical manner to the tissue sections, the results can be applied to the latter (Gagne and Miller, 1987). Fixation and embedding procedures affect the labeling efficiency and density. Fixation must be rapid to minimize antigen diffusion. This can be achieved in the case of cultured monolayers of cells and epithelial cells by immersion fixation. In certain solid tissues such as kidney and liver, fairly rapid fixation can be accomplished by vascular perfusion with aldehydes. Compared with glutaraldehyde, formaldehyde fixation generally is less damaging to antigens (Hayat, 1986). Although formaldehyde fixation causes crosslinking of globular proteins, it does not result in discernible alteration of protein secondary structure (Mason and O'Leary, 1981 ). This monoaldehyde possibly causes changes in the quaternary stucture of proteins, without apparent alterations in their secondary structure. It can be assumed that the secondary structure of proteins remains unaltered during dehydration and embedding subsequent to formaldehyde fixation. In other words, the secondary structure of proteins is 'locked in' by the process of fixation with formaldehyde. It needs to be noted that certain types of antigens (e.g. horseradish peroxidase) are preserved better with glutaraldehyde than with formaldehyde (Puchtler and Meloan, 1985; Gagne and Miller, 1987). Low concentrations of glutaraldehyde (0.1q3.5%) alone or in combination with formaldehyde (2-3%) are effective in preserving many types of antigens as well as the ultrastructure for immunogold labeling. The effect of chemical fixation on immunreactivity is very complex. For example, fixation with 0.5% glutaraldehyde alters the antibody binding capacity of each pancreatic enzyme to a different degree (Kraehenbuhl et al. 1977). Masking of antigens due to osmication should be kept in mind. All factors that affect fixation quality (Hayat, 1989) may influence the labeling density. Local orientation of the specimen and its components is also important. An alternative to chemical fixation is rapid cryofixation, since the former often decreases the immunoreactivity of antigens. Cryotechniques include freezing followed

Quantitation of immunogoldlabeling by freeze-substitution, freeze-fracturing, or freeze-drying. In these procedures, tissues or sections of them can be treated with chemical fixatives in an aqueous or gaseous state. Rapid freezing followed by freeze-substitution without any chemical fixation used in conjunction with immunogold labeling is superior to some other cryofixation procedures (Usuda et al., 1990). Tissue pieces can be rapidly frozen by metal contact freezing on the surface of a gold-plated copper block cooled with liquid nitrogen ( - 196°C), and freeze-substituted with acetone in the absence of any chemical fixative at - 80°C (Hayat, 1989). Freeze-substitution is accomplished by keeping the tissue in acetone at - 80°C for 48 h, and then the temperature is gradually raised to - 20°C. The tissue is kept at this temperature for 3 h, transferred to Lowicryl K4M, and polymerized at - 20°C with UV irradiation. The tissue is kept in the freezer at - 20°C until morrring, when thin sections are made for immunostaining. The principles governing the processes of cyrofixation and freeze-substitution are ably discussed by Kellenberger (1991). Another approach involves rapid freezing followed by thin cryosectioning and immunolabeling. This is a sophisticated procedure and requires a great deal of skill to be carried out (Tokuyasu, 1983; van Bergen en Hengouwen, 1989). Ultrarapid freezing followed by cryosectioning and cryoelectron microscopy is another advanced procedure; however, this has not been applied to immunogold labeling. Whether specimens are embedded or unembedded (frozen sections) affects the labeling density. In this respect, the advantages of thin cryosections over thin resin sections are controversial (Kellenberger and Hayat, 1991). Labeling density is also influenced whether the preembedding or postembedding approach is used. Although in the postembedding method, where the intrinsic cell matrix is supplemented by the resin, differences in labeling density across various cell structures due to penetration by the immunoreagents is less, quantitative data showing the effects of embedding on the initial cellular matrix are lacking. Significant variations in the labeling efficiency over various cell structures in Lowicryl K4M-embedded tissue sections have been reported (Griffiths and Hoppeler, 1986). The degree of resin polymerization and extent of swelling or shrinkage of not only sections but also cell organelles affect labeling efficiency. Effects of embedding media (Lowicryl K4M, LR White, JB-4, Spurr resin, and Quetol 812) on the sensitivity of immunogold labeling of membranous antigens have been studied quantitatively (Shida and Ohga, 1990). On the basis of the labeling intensity expressed as the number of protein A-gold particles per 500 nm length of the desmosomal region along the membrane, JB-4 resin sections yielded the highest labeling intensity. This intensity can be increased by pretreating the thin sections of this resin with methyl methacrylate monomer for 5 min. Such a treatment causes slight swelling of the resin, which probably facilitates the access of the antibody to the epitopes. JB-4 resin contains primarily low acid glycol methacrylate. According to Bendayan et al. (1987) also, glycol methacrylate yields the highest labeling intensity. Generally, hydrophilic resins (JB-4, LR Gold, Lowicryl K4M) yield a higher labeling intensity compared with that shown by epoxy resins (Epon). The latter contain very active epoxy groups that tend to interact with peptide groups of protein antigens. Such interactions limit the reaction between the antigen and the antibody. Epoxy resins also contain hydrophobic groups which inhibit water absorption by the section, limiting antibody access to the antigen. Acrylic resins (Lowicryls), on the other hand, possess hydrophilic groups that facilitate antibody penetration into tissue sections. The use of Lowicryl K4M also results in a lower nonspecific labeling. It should be noted that no one resin is optimal for all antigens. Immunogold labeling can also be employed for studying the eukaryotic cytoskeleton using resinless sections (Wang and Traub, 1991). However, the effects of extraction with buffers, digestion with nucleases, and removal of the embedding material with an

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organic solvent on the structure of cytoplasmic and intranuclear filaments have not been determined when using this technique. During sectioning, antigens from the section surface may wash out into the trough water. It has been shown that antigens migrate out of the section and become adsorbed to the support film of the grid (Stierhof et al., 1991). Antigens, in addition, might be mechanically transferred during sectioning. The quality of immunoreagents and reaction conditions (e.g. temperature and dilution of reagents) are also experimental variables (Slot et al., 1989b). The questionable stability of most protein-gold conjugates, especially when the protein is an immunoglobulin, should be kept in mind. The method of colloidal gold production, age, and hence possible deterioration of the probe are also relevant variables. With careful attention, these variables can be adequately controlled within one ideal experiment (Posthuma et al., 1986). They demonstrated that biochemically established relative changes in the concentration of amylase and chymotrypsinogen in secretory granules of the rat exocrine pancreas after diet treatment were quantitatively reflected in their labeling density in thin cryosections. Exogenous factors have also been discussed by Park et al. (1987, 1989) and Slot et al. (1989a,b). Endogenous factors. Endogenous factors constitute mainly the microenvironment and the condition of the antigen in the specimen. Different microenvironments in various cell compartments react differently to the exogenous factors discussed earlier. Location of the antigen in the tissue affects its accessibility to an immunoreagent. Penetration barriers such as membranes and dense cellular matrix influence the access of antigens to antibodies. The degree of compactness of the cytoplasmic matrix surrounding the antigen is probably the most important endogenous factor. Variations in the density of the matrix in which antigens are packed are serious variables in quantitative studies. Model experiments have demonstrated that the labeling density on a known amylase was maximum when it was surrounded by the gelatin matrix of less than 10% concentration; higher concentrations of gelatin hindered the penetration by the immunoreagents (Posthuma et al., 1985). However, the essence of these experiments was that at a gelatin concentration of 10% or higher, labeling was restricted to the section surface and was proportional to the antigen concentration. At a concentration below 10%, there was variable penetration and no clear proportionality. Variations in exposure of antigens to immunoreagents occur when the labeling is performed on resin sections or cryosections (Slot et al., 1989b). Penetration of gold particles is restricted to the section surface in a dense cell component (e.g. secretory granules where the antigen is at a very high concentration), whereas in the case of a less dense structure (e.g. rough endoplasmic reticulum and glycogen) they may penetrate at different levels inside a cryosection (Slot et al., 1989b). However, a consensus whether or not gold particles penetrate deeper than the section surface of a cryosection is lacking. Another important endogenous factor is the degree of steric hindrance. Steric hindrance in this case means that the presence of one antibody-gold complex physically hinders others to bind to closely located antigens (Griffiths and Hoppeler, 1986). Steric hindrance is a problem when the antigens are present in a very high concentration and very close to one another such as in viral envelopes and secretory granules. The plasma membrane and endoplasmic reticulum do not exhibit this problem. Since gold particles of a relatively large size are still used in scanning electron microscope cytochemistry, steric hindrance is a serious problem, particularly in direct procedures (Horisberger, 1989). Generally, as the size of the gold probe increases, the steric hindrance becomes acute, resulting in fewer gold particles bound to the cell surface. A number of reasons are presented to explain this problem. Each gold particle

Quantitationof immunogoldlabeling of a large size can cover many cell surface binding sites in a cluster (Horisberger, 1981). Gold particles of a large size also show slow mobility as well as high mutual electric repulsion. Mutual interpenetration of the double layers of gold particles may cause repulsion with the cell surface. Macromolecules bound to gold particles are less pliable and the marker may not interact with antigens (Horisberger, 1981). Certain binding sites due to a narrow spacing of glycoproteins on the cell suface may not be accessible to gold particles of a large size. The large size of gold particles adversely affects their diffusion even through a thin section. Some of these problems can be alleviated by using indirect methods. Effects of the size of gold particles on labeling efficiency are discussed later. Variation in penetration by the antibody-gold complex into different organelles is another important factor. Although penetration helps to increase the sensitivity of labeling, it becomes a problem for quantitative studies unless it is uniform among organelles (Girffiths and Hoppeler, 1986). One solution is to intentionally restrict the penetration to the section surface. Thus, the area of organelle exposed to the antibody-gold complexes would be proportional to the section area that it covers. This approach has been attempted by Slot and Geuze (1982). They infiltrated the tissue with polyacrylamide which is then crosslinked to form a gel that, when cryosectioned, restricts the labeling to the section surface. Polyacrylamide can be introduced with equal apparent density in every tissue compartment, so that all compartments are identical in terms of the depth of penetration of the immunoreagents in the section (Slot et al., 1989a). The problems with this approach include possible reduction in labeling sensitivity, harmful effect on antigenicity, and uncertainty whether the surface of the section is the same over various organelles. A different approach for negating the effect of different penetration rates into various organelles would be to cut very thin secions (e.g. 20-30 nm) or to use frozen cell fractions. A change in the conformation of a protein molecule during its lifetime is accompanied by variation in immunoreactivity (Slot et al., 1989a). The labeling efficiency may be different for each molecular conformation. This is exemplified by certain proteins that are known to form reversible polymers or complex with other molecules as in the case of receptors and their ligands. The obscuring effects of such variables on quantitation can be circumvented by employing monoclonal antibodies that recognize only one type of configuration of an antigen. Other factors include the possible association of the antigen with membranes or with other components. This factor varies with different microenvironments (lipids and glycoproteins) in various cell compartments. Voorhout et al. (1986) have indicated that lipopolysaccharide carbohydrate chains are responsible for the steric hindrance encountered during immunogold labeling of the pore protein PhoE of E. coli K-12. In this study ~20% more labeling was obtained by using thin cryosections compared with whole cells. This protein forms complexes with lipopolysaccharides which mask the former. On the other hand, the GaI-U mutant of E. coli K-12 with shorter carbohydrate chains showed similar labeling density on whole cells or cryosections, pointing to diminished steric hindrance. Another pitfall is encountered when the antigen is considerably smaller than the antibody molecule. Effect of the size of gold particles. The number of labeled sites represented by one gold complex (e.g. protein A-gold) largely depends on the size of the gold particle. Labeling efficiency increases with decreasing size of the gold particles within a certain size range. Yokota (1988) has indicated a six-fold increase in the labeling of catalase in liver peroxisomes when 5 nm gold particles were used compared with the labeling yielded by 26 nm gold particles. Studies by Kehle and Herzog (1987) also demonstrate the effect of the size of gold particles on the labeling efficiency. In the case of Staphylococcus aureus cell wall, the maximum number of IgG-gold (6 nm) and IgG-gold (20 nm) complexes

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bound per cell was 19,700, and 3400, repectively. The former complex represented 2.5 binding sites, while the latter corresponded to 15 binding sites. Gold particles of 1 nm in size generally yield increased efficiency with both scanning and transmission electron microscopy, although some workers believe that the size of the gold particles affects the labeling efficiency only when their size is relatively large. According to the latter view, for the 5-9 nm range used most widely, there is very little difference in the labeling efficiency; at ~ 10 nm and larger size the efficiency begins to decrease significantly. Labeling density remains unchanged when the size of the gold particles is larger than a certain size (e.g. 26 nm). One possible reason for decreased labeling density with gold particles of a large size is that the binding forces of the antibody to the antigen cannot support such particles. It should be remembered that the total labeling by protein A-gold or IgG-gold decreases with increased size of the gold particle, while the labeling affinity of the entire gold particle increases with an increase in its size to a certain degree. Simultaneous localization of two or more antigens is one of the important aspects of the colloidal gold methodology, and can be used in quantitative studies. Such labeling of multiple antigens with the same compartment, however, should be interpreted with caution in quantitative studies, since gold particles of different sizes yield different labeling efficiencies. Nevertheless, this problem, if alone, can be overcome by applying reverse labeling, i.e. by labeling antigen X with 5 nm gold particles and antigen Y with 9 nm gold particles. Subsequently, antigen X is labeled with 9 nm gold particles and antigen Y is labeled with 5 nm gold particles. Then, Xs: X 9 labeling ratio is calculated. This approach implies a remarkable monodispersed gold particle population. The labeling efficiency of gold particles of different sizes is exceedingly complex, and this topic has been discussed further by Kellenberger and Hayat (1991).

Backyround immunogold labelin9. Avoidance or at least significant reduction of nonspecific background labeling is desirable for the quantitation of immunogold labeling. A number of known and unknown factors are responsible for background labeling. Some of these factors and methods to prevent avoidable background labeling are described below. Nonspecific background labeling is caused primarily by certain components of the diluted serum. This is confirmed by carrying out experimental and control procedures identically except for the omission of primary antibody in the latter. Such a control does not show background labeling, whereas the experimental procedure exhibits considerable background labeling. To avoid this type of labeling, the use of monospecific antibodies raised in the appropriate mammalian species for the primary localization of antigens is urged. Affinity-purifiedand monoclonal antibodies generally yield specific labeling. Nonspecific binding, in some cases, can be reduced by decreasing the effective antibody concentration. In fact, antibodies should be used at their lowest effective concentration. Another method to avoid or minimize background labeling in the two-step procedure is ammonium sulfate precipitation of the IgG and/or the purification of IgG over a protein A column (D/irrenberger et al., 1991 ). The serum components that cause background labeling also possess protein A binding sites and they crystallize together with the IgG. Certain components of the serum nonspecifically bind with the polymerized resin. One approach to avoid this binding is to saturate the diluted serum with a reagent that resembles polymerized resin, thus depleting the serum components that cause background labeling by nonspecific binding with the resin (Dfirrenberger et al., 1991 ). When using Lowicryl sections, gelatin solution (0.14).2%) can be added to the blocking solutions and should be present during the entire two-step labeling procedure. Immunoglobulin G or protein A may bind nonspecifically due to protein-protein

Quantitation of immunogoldlabeling and electrostatic interactions. Such binding can be minimized by including in the rinsing and incubation steps a noncompeting protein such as BSA, fish gelatin, bovine skin gelatin, ovalbumin, or milk powder. These proteins bind to protein-reactive constitutents within the specimen and reduce electrostatic interactions by covering the exposed areas on the gold particles (Hyatt, 1989). Alternatively or concomitantly, protein A-gold complex can be incubated with the specimen. Specimens such as tissue culture cells and blood cells should be thoroughly rinsed in PBS before labeling. This is desirable since during processing some antibodies may fortuitously bind to the cell surface (Hyatt, 1989). Such antibodies, if not removed, will provide additional binding sites for protein A. The method used to prepare colloidal gold may also be the source of background labeling. Birrell et al. (1987) found that 5-nm gold particles produced by the trisodium citrate-tannic acid method showed greater nonspecific labeling than particles of similar size synthesized by other methods. Reagents used in the former method are responsible for the higher degree of background labeling. Methods for quantitation

A number of methods are in use to 'quantify' intracellular or extracellular antigens, which are briefly reviewed below. Colloidal gold-tagged antibodies are amenable to quantification by morphometric analysis of antigen density on the section surface, provided at least the following three criteria are fulfilled: (1) antigenicity survives specimen processing, (2) immunological reagents are equally accessible to the antigenic sites in different cell compartments, and (3) the immunological reagents interact with antigens in a stoichiometric relation (Kraehenbuhl et al., 1980). These goals are difficult to achieve. Presently, the most common method for quantitation is based on the principles of Weibel (1979). The quantitation is carried out in relation to the surface area occupied by the labeled cellular structure. The density of labeling is expressed as the number of gold particles counted per i.tm2 of the cell compartment. For each specimen, thin sections from at least 3-5 blocks should be analyzed. As an average, 25 electron micrographs of a constant primary magnification of 20,000 from different fields of observation should be evaluated for each antigen. The evaluation should be made by (1) counting all gold particles (Nc) falling within the labeled subcellular structures, (2) overlayering each electron micrograph with a two-dimensional lattice and then morphometrically estimating the mean area (/~m2) occupied by the labeled structures (Ac), and (3) calculating the labeling density (NA) by dividing Nc by Ac (Weibel, 1979; Mutasa, 1991). Background labeling (Nb) should be evaluated in a similar manner. The fractional surface area of each of the intracellular compartments can be measured either by an area-curvimeter or by a computer-assisted digitizer on electron micrographs enlarged, as an average, by a factor of four during printing. A computerized image analysis system has been employed for quantitation (Enestr6m and Kniola, 1990; Fishkind et al., 1990). Monoclonal antibodies and biotin streptavidin gold complex were used for quantitative analysis of immunogold labeling with the help of a semi-automatic procedure (Bonhomme et al., 1990). This procedure is a digital image analysis system. The number of gold particles per unit surface was quantified inside three cellular compartments defined in the video screen. McCaul and Williams (1990) also calculated the density of immunogold labeling over the bacterial surface by quantitative digital image analysis (Zeiss MOP-1). Other methods for quantitation include the use of latex microspheres as a reference, photometric assay, protein- [ 195Au] radiometric assay, and [ 125I]-protein-Au radiometric assay. Comparative advantages and limitations of these procedures are discussed by Kehle and Herzog (1987, 1991). Another approach for obtaining quantitative data is to correlate the ratio of labeling

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densities of two different antigens with the equivalent biochemically determined ratios (Posthuma et al., 1984). Posthuma et al. used rat pancreatic cells in which the contents of amylase and chymotrypsinogen were experimentally altered. The changes in these two protein levels were measured biochemically in cell suspension homogenates. The amylase:chymotrypsinogen ratio determined biochemically matched fairly well with that of gold particles bound to the respective proteins in immunogold-labeled thin cryosections. Thus, immunogold labeling can be relied upon as a quantitative reflection of the antigen concentration under well-defined conditions. Furthermore, fluctuations in antigen concentration in a homogeneous compartment may be measured by in situ immunogold quantitation. Attempts have been made to quantitate immunogold labeling on tissue sections using morphometric determinations (Kraehenbuhl et al., 1980; Landmann et al., 1990; Londofio and Bendayan, 1990; Desjardin et al., 1990; Yang and Gallo, 1990; Hartwig et al., 1990; Toyoda et al., 1991; Hammel and Kalina, 1991; Tak~cs et al., 1991; Girod et al., 1991 ; Berdal et al., 1991; Roux and Kan, 1991; Tanaka et al., 1991; Matsuura and Hand, 1991; Oko and Clermont, 1991; Goosen-de Roo et al., 1991; Fuhrer et al., 1991). Beesley and Betts (1985) have distinguished three poliovirus types by quantifying the immunolabeling on each virus type after immunolabeling with antisera against each of the three types. A few studies based on morphometric analysis are reviewed below. Grant and Leblond (1988) have predicted the proportion of the contents of basement membranes by counting the number of bound gold particles on a series of these membranes. The density of gold particles (expressed per [.tm2) averaged 307, 146, and 23 for laminin, collagen VI, and haparan sulfate proteoglycan, respectively. In this study the basement membrane counts were divided into two distinct parts. The 'horizontal' comparison dealt with different antibodies in a given basement membrane. Although this is not the best approach, it can be justified provided the true content of the substances is known. The 'vertical' comparison refers to the reactions to a given antibody in different basement membranes. The latter approach is more reliable, but differences in basement membrane texture and other factors limit its usefulness to tentative conclusions. The reliable approach is to use the same procedure, the same antibody applied to the same tissue under varying conditions, as for instance comparing the antibody response of the epidermal basement membrane in males and females of the same age. Quantitation of immunolabeling of nine different secretory proteins in rat exocrine pancreas was carried out by Bendayan et al. (1980). This specimen was selected because of the availability of biochemical data on enzyme protein content of various cell fractions or pancreatic juice. In this study the density of labeling was expressed as the number of gold particles counted per ].tm 2 of the cellular compartment. An increased gradient of labeling was found from the rough endoplasmic reticulum to the Golgi apparatus and zymogen granules for amylase, chymotrypsinogen, trypsinogen, carboxypeptidase A and B, and RNase. For DNase, lipase, and elastase, an increasing gradient of labeling was observed only from the rough endoplasmic reticulum to the Golgi apparatus with no further appreciable increase in the zymogen granules. A satisfactory correlation was found between the ratio of immunolabeling intensity of the various pancreatic enzymes and their corresponding ratio as determined by biochemical means (Marchis-Mouren, 1965). Controls. Control experiments are carried out to assess labeling specificity. Without a control it is difficult to be certain about a false positive labeling. If an antigen is simultaneously localized in several unrelated subcellular compartments, the labeling specificity would be suspect. Negative controls to confirm the labeling specificity of antibodies and gold probes are performed by replacing the specific antibodies with antibodies of unrelated

Quantitation of immunogoldlabeling

11

specificity and by incubating the specimens without the specific antibodies but with gold probes. Incubation with unlabeled secondary antibodies followed by gold can also be carried out. Also, the primary antibodies can be replaced by nonimmune serum from the animal in which the antibodies were raised. In enzyme-gold studies thin tissue sections should be incubated in an enzyme-gold complex, the enzymatic activity of which has been abolished by heat treatment or in the presence of a specific enzyme inhibitor. Other controls in such studies have been presented by Bendayan (1989). Antibodies directed against carbohydrate epitopes common to plant proteins may be controlled by pretreating parallel sections with periodate and HC1 (Herman, 1989). Alternatively, carbodydrate residues can be selectively removed with glycosidases. Control experiments for evaluating hybridization labeling are given by Wolber and Beals (1989). In multiple labeling studies, single labeling of each of the antigen type with respect to both labeling pattern and density is the most important control. If double labeling is carried out in sequence, the order of labeling sequence should be changed in order to verify the absence of steric hindrance and possible cross-reactivity (Namork, 1991). Both experimental and control specimens are run in parallel. The gold conjugates are checked in the electron microscope for clustering of particles. Quantitation of plasma membrane antigens In order to obtain quantitative information on the plasma membrane antigens from the numerical data generated by immungold labeling of thin cryosections, the following suggestions are in order (Howell et al., 1987). The density of label should be indicated on the basis of per micrometer of membrane length, since immunogold measurements yield the number of gold particles per micrometer of membrane profile length. This value should be combined with the section thickness to calculate the labeling density: gold particles/lxm 2 of the plasma membrane. The thickness of thin cryosections is usually 50-120 nm. The intersection counting method should be used for measuring membrane contour length. A large number of membrane areas should be sampled rather than a small area with precision. The labeling density should be related to the antigen density that has been obtained by biochemical or immunological assay, when possible. The absolute size of the compartment of interest should be determined under conditions identical to those used for labeling. Quantitation by scanning electron microscopy de Harven et al. (1987) have considered the question of the quantitative significance of counting the number of observed gold particles on the surface of a lymphocyte, using a scanning electron microscope in the backscattered electron imaging mode. In other words, do the number of observed gold particles reflect significantly the number of epitopes on the cell surface? It is easy to count the number of gold particles seen on this cell. However, only a part of the spherical cell surface is exposed to viewing, while the other part is attached on a fiat substrate and thus hidden from our view. The hidden and exposed portions of the cell surface may not be identical in terms of the concentration of a given antigen. In order for the epitopes to be labeled, they must be exposed on the cell surface. The accessibility of an epitope to antibody depends on its degree of integration in the plasma membrane (de Harven et al., 1987). The possible masking effect of surface glycoproteins needs to be evaluated in the quantitative study. The effect of fixation on antigens should also be considered. It has been pointed out that the loss of tertiary structure of proteins after fixation with glutaraldehyde is not an all or none phenomenon (Hayat, 1986). Prefixation with 0.1 o/o glutaraldehyde allows the antibody binding of 80-100% of the cell surface antigens (Van Ewijk et al., 1984). de Harven et al. (1987) also indicate that the labeling density of lymphocytes with or without

12

M.A. Hayat

prefixation is not noticeably different. Such mild fixation is necessary to preserve cell surface architecture and prevent endocytosis of the gold-ligand-receptor complex (de Harven et al., 1984). As is true in transmission electron microscopy, the use of gold particles of a small size (1-5 nm) for scanning electron microscopy results in better quantitation compared with that achieved by employing gold particles of a large size (20-40 nm). de Harven et al. (1990) have indicated that the use of 40 nm gold particles resulted in particle counts on human T lymphocytes that were approximately two orders of magnitude less than the anticipated numbers; this reduction in particle counts is due to steric hindrance. The backscattered electron imaging mode of the scanning electron microscope is preferred over the secondary electron imaging for quantitation (de Harven and Soligo, 1989). The former provides unambiguous detection of the gold probe, and the topographical distribution of labeled antigens can be correlated with the cell surface morphology. The reason for this advantage is that backscattered electron imaging shows differences in average atomic number which are sufficiently large between the metallic gold and unlabeled cell structures as well as the carbon coat. Gold labeling can also be imaged with electronically mixed secondary and backscattered signals (de Harven et al., 1984). Quantitative methods for backscattered detection of colloidal gold markers have been reported (Stump et al., 1988; de Harven and Soligo, 1989). The development of a high resolution field emission scanning electron microscope has permitted the detection of 4-20 nm colloidal gold particles on cell surfaces coated with carbon by secondary electron emission (Albrecht et al., 1988). This instrument also allowed the imaging of 5 15 nm gold particles by backscattered electron imaging even though the specimens were coated with 1-9 nm thick platinum layer (Erlandsen et al., 1990a). The development of an improved single crystal (YAG-Ce type) detector for backscattered electron imaging by Autrata (1989) has permitted the visualization of 1 nm gold particles at accelerating voltages as low as 3-5 kV (Erlandsen et al., 1990b). Such low voltages significantly lessen the radiation damage to the specimen. Although, as stated above, backscattered electron imaging is superior to secondary electron imaging, the yield of the former electrons is very small for thin specimens at high accelerating voltages of the STEM (Otten, 1990). This problem results in a weak signal which is too noisy for detecting small gold particles. This limitation can be circumvented by using high-angle annular dark-field imaging (Pennycook, 1989). This imaging detects atomic number contrast with very high sensitivity. The electrons used are diffracted to high angles (>5 °) for imaging with the annular dark-field STEM detector. Acknowledgement--This work was supported in part by a Released Time for Research granted by Kean College.

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Bendayan, M., 1984. Enzyme-gold electron microscopic cytochemistry: a new affinity approach for the ultrastructural localization of macromolecules. J. Electron Microsc. Tech., 1: 349. Bendayan, M., Nanci, A. and Kan, F. W. K., 1987. Effect of tissue processing on colloidal gold cytochemistry. J. Histochem. Cytochem., 35: 983. Bendayan, M., 1989. The enzyme-gold cytochemical approach: a review. In: Colloidal Gold: Principles, Methods, and Applications, Hayat, M. A. (ed.), Academic Press, San Diego, Vol. 1, pp. 117-147. Benhamou, N., 1989. Preparation and application oflectin-gold complexes. In: Colloidal Gold; Principles, Methods, and Applications, Hayat, M. A. (ed.), Academic Press, San Diego, Vol. 1, pp. 95-143. Berdal, A., Nanci, A., Smith, C. E., Ahluwalia, J. P., Thomasset, M., Cuisinier-Gleizes, P. and Mathieu, H., 1991). Differential expression of calbindin-D 28 kDa in rat incisor ameloblasts throughout enamel development. Anat. Rec., 230: 149. 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