ARTICLE |
Correspondence to: Peter R. Cook, Sir William Dunn School of Pathology, Univ. of Oxford, South Parks Road, Oxford OX1 3RE, UK. E-mail: Peter.Cook@Path.OX.AC.UK..
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Summary |
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Some SR proteins are associated with eukaryotic transcripts as they move from synthetic sites (transcription "factories"), through downstream sites, to nuclear pores. Downstream sites can also be isolated as large nuclear ribonucleoprotein particles of ~200 S (diameter ~50 nm). In ultrathin sections of HeLa nuclei, indirect immunogold labeling with a specific antibody gives many small clusters of ~10 gold particles (diameter 5080 nm). We gauged errors in estimating the diameter of underlying structures marked by immunogold probes (lengths ~20 nm). We examined systematically how probe dimensions affected cluster diameter. Probes contained one to three immunoglobulin molecules, sometimes a protein A molecule, and a gold particle of 515 nm. We found that (a) immunolabeling particles were tightly packed, (b) reducing particle size by 5 nm reduced cluster diameter by 10 nm, (c) reducing the number of immunoglobulins in the immunolabeling sandwich from three to two reduced cluster diameter by ~4 nm, (d) replacing the last immunoglobulin in a sandwich with protein A increased diameter by ~7 nm and led to a peripheral concentration of particles, and (e) increasing the number of layers in the sandwich increased sensitivity. Assuming that underlying structures had diameters of 50 nm, we find that errors ranged from -20% to +50%. (J Histochem Cytochem 46:985992, 1998)
Key Words: immunoelectron microscopy, immunogold labeling, LR White, resolution
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Introduction |
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Antibodies attached to gold particles are widely used to mark antigens lying on the surface of sections. The antibody binds specifically to the antigen and the dense gold particle can be seen easily in the electron microscope (
We illustrate the problem using a specific example, SR sites in the nucleoplasm of mammalian cells. These sites are defined using a monoclonal antibody (MAb) that recognizes a phosphorylated subset of the SR family of proteins (
Several interrelated factors complicate analysis. First, SR sites with diameters of ~65 nm are not much larger than the smallest probe in everyday use (i.e., an immunoglobulin with a length of 9 nm conjugated with a 5-nm particle). Second, they can be detected efficiently only by using two or more antibody layers, but then the probe has a size close to that of the site under analysis. Third, sites are so small that they become saturated with ~10 immunolabeling particles. Analysis must be conducted at or close to saturation levels, because slight reductions inevitably lead to a significant underestimate of diameter. This follows because halving the number of particles over a structure from a saturation level of 10 to 5 has a significant effect on the area occupied by the cluster. Fourth, stereological analysis should (but usually does not) take into account the probabilistic effects of dealing with so few particles. We correlated changes in the number of layers in the immunolabeling sandwich and size of gold particle with the observed diameter of the cluster. We also investigated how replacement of one immunoglobulin in the sandwich with protein A affected labeling. We hope that these results will be of general use to others trying to gauge errors in estimating the diameter of underlying structures using probes with dimensions close to that of the structure being analyzed.
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Materials and Methods |
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Immunolabeling
Phosphorylated SR antigens were detected using an MAb produced by cell line m104 (ATCC CRL-2067; Rockville, MD) (
For Figure 1, suspension cultures of HeLa cells were pelleted, washed with PBS, and fixed (10 min, 0C) in 4% paraformaldehyde in 250 mM Hepes (pH 7.4) and postfixed (50 min; room temperature) in 8% paraformaldehyde in 250 mM Hepes. After washing successively in PBS, 0.02 M glycine in PBS, and then PBS, cells were dehydrated in ice-cold ethanol, embedded in LR White (polymerization 5 hr at 56C; London Resin Company, Reading, Berks, UK) and ultrathin sections on nickel grids immunolabeled. Unspecific binding was blocked by preincubation (30 min) in PBS (pH 8.2) with 0.1% Tween 20 and 1% BSA (PBTB buffer). Next, sections were incubated (2 hr) with the anti-SR MAb (1:100 dilution of supernatant) in PBTB, washed in PBS (pH 8.2), and incubated with secondary and tertiary layers as follows. For Ig:Ig:5 sandwiches, sections were incubated (1 hr) with goat anti-mouse IgG conjugated with 5-nm gold particles (1:50 dilution) in PBTB. For Ig:Ig:Ig:Au sandwiches, sections were incubated (1 hr) with rabbit anti-mouse IgG (1:50 dilution) in PBTB, rewashed, and incubated (1 hr) with goat anti-rabbit IgG conjugated with 5-, 10-, or 15-nm gold particles (1:50 dilution) in PBTB. For Ig:Ig:pA:Au sandwiches, sections were incubated (1 hr) with rabbit anti-mouse IgG (as above), rewashed, and incubated (1 hr) with protein A conjugated with 5- or 9-nm gold particles (1:100 dilution) in PBTB without Tween. After binding gold particles, sections were washed in PBS and then with water, dried, and contrasted with a saturated solution of uranyl acetate in 70% ethanol. Sections were observed in a Zeiss 912 Omega electron microscope (accelerating voltage 80 kV) and images collected using a CCD camera (1024 x 1024 chip; depth 14 bits).
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For Figure 4, polystyrene beads (diameter 1 µm) coated with biotin (Sigma; Poole, Dorset, UK) were embedded in 2% agarose (type VII; Sigma), dehydrated in ethanol, embedded in LR White (polymerization 5 hr at 56C), and ultrathin sections immunolabeled using one to three layers as described above. For one layer, the first antibody was goat anti-biotin conjugated with 5- or 10-nm particles (1:100 dilution). For two layers, the first antibody was goat anti-biotin (1:100 dilution) and the second rabbit anti-goat IgG conjugated with 10-nm particles (1:50 dilution). For three layers, the first was goat anti-biotin (1:100 dilution), the second rabbit anti-goat IgG (1:50 dilution), and the third protein A conjugated with 9-nm particles (1:100 dilution).
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Image Analysis
Images such as those in Figure 1 were analyzed as follows ((xy), and mean cluster diameter,
from the number of clusters (N) and the diameter of individual clusters (D1, D2,...DN) using
(d) calculation of the "shape factor" of clusters from 4 (area/perimeter2). Values lay between 0.640.79, indicating that the underlying structures were close to spheres (which have a value of 1); (e) measurement of positions of individual particles in a cluster, the center of gravity of the cluster, and distance to nearest neighbors. [Note that use of the "new" stereology here is inappropriate because the structures analyzed have diameters less than the section thickness (for review see
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Results |
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The Problem
Figure 1A illustrates the problem we wish to solve. HeLa cells were fixed and embedded in LR White. After preparation of conventional ultrathin sections, sites on the surface of sections that contained SR antigens were indirectly immunolabeled with gold particles. The immunolabeling sandwich contained a primary anti-SR antibody, a secondary immunoglobulin (Ig), and a third Ig conjugated with 10-nm gold particles. For convenience, we call such a sandwich Ig:Ig: Ig:10. [The corresponding three-layer sandwich containing protein A conjugated with a 9-nm gold particle will be called Ig:Ig:pA:9.] Immunolabeling gold particles are found in clusters over the nucleus. We define a cluster as a group of 2 particles lying within 40 nm of each other for quantitative analysis. Some electron-dense material underlies the clusters. This is not seen without immunolabeling and is due to the high concentration of protein in the immunolabeling sandwich. Ideally, we wish to deduce the true diameter of the underlying structures containing SR antigens by analyzing the clusters of gold particles. In practice, we do not even know whether the observed diameter of the clusters is smaller or larger than the true diameter, and we would like to obtain some estimate of the error. We will assume here that the true diameter is 50 nm (see Discussion).
The primary antibody used to detect SR sites is a mouse IgM. This primary antibody can be detected using a secondary antibody raised against either an IgM or an IgG (because such antibodies crossreact with IgMs). In principle, an anti-IgM should give higher detection levels than a crossreacting anti-IgG. However, in practice, two experiments showed that detection levels were equivalent. In the first, the primary anti-SR antibody was adsorbed onto a grid and immunodetected using 10-nm gold particles conjugated either to an anti-IgM or an anti-IgG. The two secondary antibodies gave equivalent labeling intensities (not shown). In the second experiment, SR sites in sections were detected using the two secondary antibodies, again with equivalent results (Table 1; compare Methods 4 and 5). Therefore, we used an anti-IgG as the second antibody for most experiments, because results obtained with it are of wider interest.
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SR clusters like those seen in Figure 1A have major and minor (orthoganol) axes of 61 ± 13 nm and 51 ± 11 nm (Table 1). Because sections are cut randomly, few underlying structures present profiles with maximal diameters to the immunolabeling probe. We can calculate a maximal diameter of 64 nm by correcting for this "Holmes" effect using standard stereological methods, if we assume that the underlying structures have similar major and minor axes (see Materials and Methods; reviewed by
The Effect of Number of Immunolabeling Layers and Particle Size
Because the estimated diameter was larger than the assumed diameter of the structure, we must correct for particle size and probe length. Therefore, we investigated the effects of reducing both, and typical examples are illustrated in Figure 1BG. Reducing particle size slightly reduced cluster diameter; extrapolation back to zero particle size (for both Ig:Ig:Ig:Au and Ig:Ig:pA:Au sandwiches) gave cluster diameters of 42 nm (Figure 2). The slope of the line for Ig:Ig:Ig:Au indicates that particle diameter is directly related to cluster diameter, with a reduction in particle diameter by 5 nm reducing cluster diameter by ~10 nm (consistent with a reduction of 5 nm on each side). However, reducing the number of immunoglobulins bridging antigen and gold particle from three to two had less of an effect (consistent with a reduction of only 2 nm on each side) (Figure 1E; Table 1). Unfortunately, use of only one immunoglobulin in the bridge gave too little labeling for accurate analysis (not shown, but see below). Perhaps surprisingly, replacing the last immunoglobulin (length ~9 nm) with a shorter protein A (pA) molecule (diameter ~5 nm) (
When we used protein A, we noticed that gold particles were often peripherally distributed in a cluster (Figure 1F and Figure 1G). Images gave the impression that long immunolabeling complexes had first bound to clustered antigens on the surface of the grid before they fell outwards to leave a circle of gold particles around the edge. Quantitative analysis confirmed this peripheral distribution (Figure 3). The position of each of 600 particles was measured relative to the center of gravity of a cluster; these relative positions are plotted in Figure 3. Ig:Ig:Ig:10 complexes gave a uniform distribution in both a scatter plot and a histogram, in which particle densities in successive 4-nm rings were plotted (Figure 3B). However, Ig:Ig:Ig:5 complexes showed a slight tendency to be located peripherally (Figure 3A), whereas complexes containing protein A were obviously concentrated there (Figure 3C and Figure 3D).
Immunolabeling Particles Are Tightly Packed
A protein coat surrounds the gold particle used in the final layer of an immunolabeling sandwich. In general, protein A gives a thicker coat than an immunoglobulin, small particles are surrounded by thicker layers, and thickness depends on the protein concentration present during binding. For the size of particles used here, layers are 36 nm thick (
Effect of Layers on Sensitivity
Both logic and the above results suggest that it is sensible to use the smallest probe possible (i.e., one immunoglobulin conjugated with a small gold particle). In practice, however, increased resolution comes at the price of reduced sensitivity (
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Discussion |
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Problems Associated with Determining the Size of SR Sites by Immunolabeling
SR sites are defined using an MAb that recognizes a phosphorylated subset of the SR family of proteins (
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The True Size of SR Sites
Most SR sites are easily extracted from nuclei (Iborra et al. in press), and they can then be isolated on sucrose gradients as large nuclear ribonucleoprotein particles of ~200 S (30% when dehydrated, a necessary part of both procedures (for reviews see
To establish the true diameter of SR sites, we determined how variations in the number of immunoglobulin (or protein A) molecules in the immunolabeling sandwich, and the size of the gold particle, affected cluster diameter. We can draw several conclusions from these studies.
First, as expected, reducing particle size reduces cluster diameter (Figure 5A and Figure 5B). Extrapolation back to zero particle size (for both Ig:Ig:Ig and Ig:Ig:pA sandwiches) gives cluster diameters of ~42 nm (Figure 2). This value is less than the 50 nm measured by negative staining, perhaps because of effects such as those illustrated in Figure 5F. A reduction in particle diameter by 5 nm reduces cluster diameter by ~10 nm; this is consistent with a reduction of 5 nm on each side, and so with the model in Figure 5A. Then, we would expect that reducing the number of molecules bridging antigen and gold particle would also reduce cluster diameter, but it had little effect (Figure 1F and Figure 2). However, the two results can be reconciled if complexes tend to be arranged as in Figure 5B, with peripheral particles on the outside of peripheral immunolabeling complexes.
Second, reducing the number of immunoglobulins in the immunolabeling sandwich from three to two reduced cluster diameter by ~4 nm (Table 1).
Third, under our conditions immunolabeling particles are tightly packed. A protein "halo" of 36 nm surrounds the gold particle used in the final layer of the sandwich; halo thickness is inversely related to particle size, with protein A forming a thicker coat than an immunoglobulin (
Fourth, and perhaps surprisingly, replacing the last immunoglobulin (length ~9 nm) in the sandwich with a protein A molecule (diameter ~5 nm) slightly increases cluster diameter (Figure 1G and Figure 1H; Table 1). With protein A, gold particles are often concentrated at the periphery (Figure 1G, Figure 1H, and Figure 3), as if long complexes fall outwards after binding. This can be explained by the lower valency of protein Agold complexes. Although an individual immunoglobulin and protein A molecule have roughly equivalent numbers of binding sites when bound to a gold particle (
Fifth, and as expected, increasing the number of layers in the sandwich increases sensitivity (Figure 4; Table 2). Unfortunately, four layers generally give too high a background for general use, and one layer, which should give the highest resolution, is not sufficiently sensitive to give acceptable levels of labeling (not shown).
Which is the best approach to use to determine the diameter of an SR site? Clusters of Ig:Ig:5 complexes fortuitously give a diameter of 50 nm (Table 1), which we assume is the correct one (but see above). In practice, the higher sensitivity of three-layered sandwiches is required to detect most sites, whereas larger particles of 9 or 10 nm are more easily seen at a conveniently low magnification.
Corrected Diameters and Numbers of SR Sites
Using Ig:Ig:pA:9, we found previously that SR sites had diameters of 76 nm (Iborra et al. in press). If the diameter of an SR site is truly 50 nm, this means that we overestimated diameter by ~50%. We also calculated the total number of sites in 3-D space (using standard stereological procedures) from the numbers and diameters of clusters seen in 2-D sections, with knowledge of nucleoplasmic volume (e.g.,
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Acknowledgments |
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Supported by the Wellcome Trust.
We thank Jeremy Sanderson for his help.
Received for publication January 21, 1998; accepted April 8, 1998.
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