SYMPOSIUM |
Correspondence to: Moïse Bendayan, Dept. of Pathology and Cell Biology, Université de Montréal, CP 6128 Succ. Centre Ville, Montreal, Quebec H3C 3J7, Canada. E-mail: moise.bendayan@umontreal.ca
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Summary |
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Amplification of immunological signals with catalyzed reporter deposition (CARD) allows improved detection of scarce tissue antigens in light and electron microscopy. The technique takes advantage of the oxidation ability of horseradish peroxidase (HRP), in the presence of hydrogen peroxide, to yield the accumulation of one of its specific reporter-tagged substrates. This immunocytochemical approach continues to be improved by the introduction of new reporter molecules tagged to tyramine or to other HRP substrates. In this study we introduced a novel HRP substrate tagged to Nanogold particles. The amplification protocol is based on the application of a specific primary antibody, a biotinylated secondary antibody, streptavidinHRP, and an HRP substrate coupled to Nanogold, followed by silver intensification. In addition to amplification of immunological signals of high resolution, direct accumulation of Nanogold particles at target sites by enzymatic activity of HRP improves the efficiency of the technique compared to other amplification protocols. Moreover, this approach combines the CARD amplification potentials with the ultrasmall gold probe and the silver intensification method. Immunolabeling obtained by light and electron microscopy, as well as immunodot assay using this new amplification strategy, appear to be highly sensitive, specific, and of enhanced intensity. (J Histochem Cytochem 48:461469, 2000)
Key Words: CARD, Nanogold, immunocytochemistry, immunodot assay, silver enhancement
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Introduction |
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THE CATALYZED REPORTER DEPOSITION approach (CARD) is now widely used in cell biology for membrane immunoassays, Western blotting, in situ hybridization, and immunocytochemistry (
To expand the possibilities of this approach we have now developed a novel probe, the HRP substrateNanogold, which takes advantage of the small 1.4-nm Nanogold particles and of the silver enhancement protocol. Both the Nanogold and the silver enhancement contribute to higher labeling intensities. Small gold particles such as those of 15-nm colloidal gold and Nanogold are known to give higher labeling densities (
In this study we used three different immunolabeling approaches in light and electron microscopy to assess the amplification obtained with a novel HRP substrateNanogold probe. The simple indirect streptavidinNanogold protocol was compared to biotinylLC-LCtyramide/streptavidinNanogold and to HRP substrateNanogold. The results obtained with the latter approach appear superior in allowing the immunolocalization of low amounts of antigenantibody complexes. Moreover, the CARDNanogold amplification system was assessed on membrane immunoassay and was proved to be quite sensitive.
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Materials and Methods |
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Preparation of a Novel HRP SubstrateNanogold
A new HRP substrate consisting of ligands other than tyramine was covalently coupled to a 1.4-nm Nanogold-type gold cluster according to previously described techniques (
Immunocytochemistry
For light microscopic immunocytochemistry, fragments of normal rat pancreas were fixed in Bouin's fixative and embedded in paraffin according to standard techniques. Five-µm-thick sections were mounted on glass slides, deparaffinized, and rehydrated through decreasing concentrations of ethanol. After washing in 0.01 mol/liter PBS, pH 7.2, the sections were incubated with 3% H2O2 in PBS for 10 min to quench endogenous peroxidase activity. The sections were then briefly washed in PBS and incubated with the specific guinea pig anti-porcine insulin antibody (ICN; Costa Mesa, CA) diluted 1:200 or 1:10,000 in PBS for 2 hr at room temperature (RT). The tissues were washed twice for 5 min in PBS and once for 5 min in 0.15 mol/l NaCl, 0.05% Tween-20 (SigmaAldrich Canada; Oakville, Ontario, Canada), 0.1 mol/liter Tris-HCl buffer, pH 7.5 (TrisTween buffer), before incubation in Tris1% bovine serum albumin (BSA) solution for 30 min. Then the biotinylated goat anti-guinea pig IgG antibody (GAG-B; Jackson Immunoresearch Laboratories, West Grove, PA) diluted 1:500 in Tris, pH 7.5, containing 0.5% BSA (TrisBSA) was applied to the sections for 1 hr at RT. After three washes of 5 min each in TrisTween buffer, the tissue sections intended for the indirect simple labeling protocol were incubated for 30 min with streptavidinNanogold (Nanoprobes) diluted 1:500 in Tris buffer. The sections intended for the amplification protocol were incubated for 30 min with streptavidinHRP (NEN Life Science; Boston, MA) diluted 1:500 in TrisBSA, washed in TrisTween buffer for 15 min, and incubated for 10 min with the HRP substrateNanogold complex diluted 1:50 in 0.05 mol/liter TrisHCl, pH 7.5, containing 0.03% H2O2. The sections processed for simple and amplified protocols were then washed for 15 min in TrisTween buffer and rinsed several times in ultrapure water. Enhancement of the Nanogold was performed by incubating the slides in the LI Silver enhancement solution (Nanoprobes) for about 2030 min. For each of the protocols, the goldsilver development was allowed to proceed until reaching an optimal signal: maximal specific reaction with low levels of background. Sections were then thoroughly rinsed with water for 5 min, mounted in PBS50% glycerol without counterstaining, and observed under a Leitz Orthoplan microscope (Leica Microsystems; Montreal, Quebec, Canada).
For the electron microscopic immunocytochemical study, small fragments of normal rat pancreas were fixed by immersion with 0.1 mol/liter phosphate-buffered 1% glutaraldehyde for 2 hr at RT, washed in the same buffer, dehydrated in methanol, and embedded in Unicryl at -25C as described previously (
The biotinyllong chain-long chaintyramide (biotinyl-LC-LC-tyramide) protocol was also tested for its amplification properties. For a fair comparison, streptavidinNanogold and silver enhancement were used as the detection step for the deposited biotin molecules. For the tyramide amplification protocol in electron microscopy, we followed the previously described technique (
The specificity of the immunolabelings was assessed by several control experiments, including antigen adsorption, omission of the primary or secondary antibody, omission of the streptavidinHRP reagent, or omission of the HRP substrateNanogold.
Immunodot Assay
Normal rabbit IgGs were obtained from Jackson Immunoresearch (Bio/Can; Mississauga, Ontario, Canada) and serial dilutions in PBS were made from a starting concentration of 10 µg/ml down to 0.001 µg/ml. One µl of each solution was spotted on a strip of nitrocellulose membrane (0.45 µm) (Gibco BRL; Burlington, Ontario, Canada) and allowed to dry for 30 min. The membrane was then blocked for 60 min with 5% BSA in PBS containing 0.05% Tween (PBST), incubated with the biotinylated anti-rabbit IgG diluted 1:500 with 1% BSA in PBST for 2 hr at RT, and washed for 15 min with PBST buffer. The strip was incubated with streptavidinHRP (1:500 in PBST containing 1% BSA) for 30 min at RT, washed in PBST buffer for 15 min, rinsed in Tris, and amplified using the HRP substrateNanogold complex diluted 1:200 in 0.05 mol/liter TrisHCl, pH 7.5, containing 0.03% H2O2 for 10 min at RT. After washing with TrisTween buffer and deionized water, the nitrocellulose membrane was treated with the LI Silver enhancement solution for 30 min, washed in water, and air-dried. For control, the same protocol was followed with one exception, the omission of the incubation with the HRP substrateNanogold.
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Results |
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In our comparative study of the CARDNanogold amplification system by light and electron microscopy, we first applied the simple protocol, making use of the Nanogold coupled to a species-specific secondary antibody. Amylase antigenic sites in pancreatic acinar cells were revealed. In electron microscopy, this simple indirect labeling technique along with a rather diluted primary antibody, followed by silver enhancement, resulted in a particulate black signal over the different cellular compartments involved in protein secretion. With the anti-amylase antibody diluted 1:200, specific labeling but of low intensity was present over the rough endoplasmic reticulum, the Golgi apparatus, the condensing vacuoles, and the secretory granules (Fig 1A). For the amplification protocols, using the same antibody dilution, the biotinyllong chain-long chaintyramide probe combined with the streptavidinNanogold reagent and silver enhancement led to higher intensities of labeling over structures containing amylase. However, some background staining was obtained over nuclei and mitochondria (not shown). To improve the results, a novel signal amplification protocol was developed using a new probe, the HRP substrateNanogold. This HRP substrateNanogold protocol consists of direct and specific deposition of Nanogold on its reaction with peroxidase. Immunolocalization of amylase in the acinar cells using this novel approach led to significant increases in signal compared to that obtained with the simple approach. Electron-dense particulate deposits in high numbers were present over cellular compartments containing amylase (Fig 1B). This labeling was associated with low background staining over nuclei and mitochondria. Moreover, application of this protocol with a more concentrated primary antibody led to very high labeling intensities detectable at low magnification and allowing a general overview of the labeled tissue (Fig 1C). Despite the lack of counterstaining, the silver-intensified reaction product permitted clear outlining of the labeled structures. The dark labeling present over the RER, the cisternae of the Golgi apparatus, the condensing vacuoles, and the secretory granules made possible a high definition of amylase-secreting cells and organelles at low magnification (Fig 1C). In addition, nuclei and mitochondria showed very little labeling, demonstrating the overall high specificity of the reaction.
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By light microscopy, immunodetection of insulin antigenic sites in rat pancreatic islet cells revealed a strong positive signal when the primary antibody was used at 1:200 dilution with both the simple (streptavidinNanogold) and the amplified (HRP substrateNanogold) immunolabeling technique (Fig 2A and Fig 2C). The staining appeared as a black precipitate localized over the major part of the islets of Langerhans, contrasting with the surrounding acinar cells devoid of labeling. Differences between the simple and the amplified technique were more apparent when the primary antibody against insulin was used at much higher dilutions. The use of the anti-insulin antibody at 1:10,000 dilution, in combination with the simple indirect labeling technique (streptavidinNanogold) and silver enhancement, resulted in lack of staining over the pancreatic islets (Fig 2B). In contrast, the same highly diluted antibody in combination with the HRP substrateNanogold was able to generate a positive specific result. Black staining was observed over the insulin-secreting cells of the pancreatic islets, with no background over the exocrine parenchyma (Fig 2D). Furthermore, the same protocol at the electron microscopic level supported those results (Fig 3A and Fig 3B). With the primary antibody diluted at 1:200, very intense labeling, revealed by the silver-enhanced gold particulate marker, was obtained over the dense core of the insulin secretory granules, with almost no diffusion of the reaction product into the halo of the granules (Fig 3A). Moreover, a similar positive labeling was also obtained with the anti-insulin diluted 1:10,000 (Fig 3B).
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The specificity of the labelings obtained with both the simple and the amplified technique in light and electron microscopy was confirmed by the control experiments. Addition of the corresponding antigens to the antibodies or omission of the primary antibody abolished the labeling. Other control experiments, such as omission of the biotinylated secondary antibodies or omission of the streptavidinHRP reagent, also led to absence of labeling (Fig 4A). However, protocols in which the HRP substrateNanogold reagent was omitted but otherwise completed with the streptavidinHRP step and the silver intensification procedure gave a positive signal of low intensity (Fig 4B).
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Determination of the sensitivity of the CARDNanogold amplification method was evaluated by an immunodot assay with known concentrations of protein. Silver deposits that revealed the proteins bound on the nitrocellulose membrane were still detectable at protein concentrations of 0.005 and 0.001 µg/ml. Because only 1 µl of these dilutions was spotted on the membrane, the positive reactions show that an amount of protein between 5 and 1 pg can be detected with this amplification technique (Fig 5). However, as with the control experiment involving omission of the HRP substrateNanogold reagent in immunoelectron microscopy (Fig 4B), the silver intensification procedure was able to reveal the HRP molecules on immunodots (not shown). Nevertheless, the sensivity of the reaction was much lower than with the amplification procedure.
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Discussion |
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Back in the early 1990s the CARD technique was essentially based on the deposition of reporter molecules, such as biotin and FITC, through the reaction of HRP on its substrate tyramide. It has since been extended to various other reporter molecules (
The present work demonstrates that the CARDNanogold system can be employed successfully as a highly sensitive, high-resolution immunoenzymatic technique for light and electron microscopy. The approach requires five successive incubation steps with (a) the specific primary antibody, (b) the corresponding biotinylated secondary antibody, (c) the streptavidinHRP, (d) the HRP substrateNanogold complex, and finally (e) the silver enhancement. The results obtained with the amplification protocol at the electron microscopic level support the fact that accumulation of labeled substrate allows a single antigenantibody complex to be revealed by multiple reporter molecules (
On the other hand, we have also demonstrated an increase in the detection limit of antigens with the use of the HRP substrateNanogold probe. For this purpose, a highly diluted antibody against insulin was used to simulate conditions in which antigenantibody complexes are present in very small numbers. Incubation of pancreatic tissue with the anti-insulin antibody diluted 1:10,000, combined with the simple indirect protocol, resulted in an absence of signal. In contrast, the amplification protocol yield specific positive staining in both light and electron microscopy, making it possible to detect the signal generated by only few antigenantibody complexes.
The amplification capabilities of the CARD approach combined with the small Nanogold probe, the silver enhancement, and a rather concentrated antibody (anti-amylase 1:10; Fig 1C) yield a very strong labeling over the pancreatic cells. Owing to this very intense signal, labeled cellular compartments could be detected at low magnification in electron microscopy without any counterstaining. This protocol, used with moderately concentrated antibodies, could be very useful for the screening of immunostained tissue sections or even for labeled subcellular structures. Observations were further facilitated by the very low background found over nuclei and mitochondria. In what concerns the specificity of the results, this was confirmed by the control experiments. However, staining of low intensity was observed with the control protocol involving the omission of the HRP substrateNanogold, either by immunoelectron microscopy or by immunodot. One explanation for this low signal, which indeed appeared specific, could be the silver intensification of the hemic iron atom present in the peroxidase molecule. In this case, the metallic atoms may have been transformed into iron sulfides or selenides because, with only few exceptions, endogenous or exogenous metals in tissues must be present under these forms to be able to react with the silver developer (
Sensitivity of the CARDNanogold amplification method was assessed with an immunodot assay with known concentrations of protein. Serial dilutions of normal rabbit IgG spotted on membrane down to 1 pg of protein were revealed. This result demonstrates a detection limit comparable to that of ELISA, enhanced luminol luminescence, and to the one-cycle amplification with biotinyltyramide and the two-cycle amplification with fluoresceintyramide visualized with the streptavidinHRPDAB system used by
The major drawback of the CARDNanogold technique relates to the silver enhancement step in its use for transmission electron microscopy. The homogenous 1.4-nm gold particles are difficult to visualize, and silver intensification is essential to increase their size. However, the silver enhancement process leads to a heterogeneity in the size of goldsilver particles, which hampers quantification of immunolabeling. The disparity in particle size may arise from the local gold label density that affects the percentage of enlarged particles and the final particle size. In addition, large particles might result from fusion of small ones, making quantification of immunolabeling density problematic and leading to underestimations (
The new HRP substrateNanogold probe, used as an amplification/detection step in our CARD protocol, leads to high-sensitivity immunolabeling. Moreover, it pushed back the detection limit afforded by the simple indirect Nanogold protocol. The combination of three amplification methods, i.e., the CARD protocol, the use of the small Nanogold probe, and the silver intensification technique, has provided improved, very sensitive, and intense immunolabeling by both light and electron microscopy.
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Footnotes |
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Presented in part at the New Frontiers in Gold Labeling Symposium, 5th Joint Meeting of the Japan Society of Histochemistry and the Histochemical Society, University of CaliforniaSan Diego, July 2326, 1998.
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Acknowledgments |
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Supported by grants from the Medical Research Council of Canada and the US National Institutes of Health. GM is recipient of a studentship award from the Medical Research Council of Canada.
We thank Dr Frederic R. Furuya of Nanoprobes for his aid in the preparation of the HRP substrateNanogold. We also thank Mr Jean Léveillé for photographic work.
Received for publication November 27, 1999; accepted December 1, 1999.
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