ARTICLE |
Correspondence to: Cornelis J.F. Van Noorden, Dept. of Cell Biology and Histology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. E-mail: c.j.vannoorden@amc.uva.nl
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Summary |
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Signal amplification techniques greatly enhance the sensitivity of immunohistochemical (IHC) and in situ hybridization (ISH) methods. In particular, catalyzed signal amplification (CSA) using labeled tyramide or Nanogoldsilver staining is an important signal amplification tool. We have applied a combination of both techniques, as has been introduced for ISH, for a further increase in sensitivity of an IHC method to detect cathepsin B. This lysosomal proteinase can also be expressed extracellularly, particularly in relation to cancer metastasis. Higher sensitivity of the IHC method was needed because existing methods failed to demonstrate cathepsin B protein where cathepsin B activity was found with a fluorescence enzyme histochemical method. Combined CSA and Nanogoldsilver staining provided the sensitivity that was required. Moreover, this signal amplification method enabled the use of a 10-fold lower concentration of primary antibody (1 µg/ml). Nonspecific background staining was low provided that endogenous biotin, avidin, and peroxidase were completely blocked. The method was reproducible when all steps, and particularly the silver enhancement step, were rigidly controlled. The method resulted in localization patterns of cathepsin B protein that were in agreement with those of cathepsin B activity in serial sections of rat liver containing colon cancer metastases. We concluded that combined application of CSA and Nanogoldsilver staining provides high sensitivity for immunohistochemical methods and that activity localization by an enzyme histochemical method is a very attractive alternative to IHC localization of an enzyme because it is at least as sensitive, it is rapid and simple, and it provides direct information on the function of an enzyme. (J Histochem Cytochem 48:933941, 2000)
Key Words: tyramide, signal amplification, Nanogold, immunohistochemistry, enzyme histochemistry, proteinase, cathepsin B, colon cancer metastasis, liver, posttranslational processing
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Introduction |
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The sensitivity of immunohistochemical methods has been increased enormously in recent years by the development of antigen retrieval methods (
Recently, we compared localization patterns of protein and activity of cathepsin B, a lysosomal proteinase, during cancer progression in human colon (
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Materials and Methods |
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Cancer Cells and Induction of Liver Metastases
A colon adenocarcinoma cell line, CC531s, was obtained from a moderately differentiated and weakly immunogenic colon adenocarcinoma after experimental induction in WagRij rats by treatment with 1,2-dimethylhydrazine (
Preparation of Liver Tissue Sections
Livers were removed immediately after sacrifice at 3 weeks after administration of the cancer cells and the individual liver lobes were divided and cut into small pieces (up to 5 mm thick). The pieces of liver that contained macroscopically visible metastases were frozen in liquid nitrogen and stored at -80C until further use. Sections (10 µm thick) were cut on a motor-driven cryostat with a rotary retracting microtome (5000 M; Microm, Walldorf, Germany) at a constant speed and a cabinet temperature of -25C. Sections were mounted on glass slides coated with Biobond (Electron Microscopy Sciences; Ft Washington, PA) and stored at -20C until use.
Immunohistochemical Procedures
Cryostat sections were taken from the -20C storage cabinet and were immediately fixed in 3.7% paraformaldehyde (20 ml) supplemented with 100% acetic acid (10 ml) and 100% ethanol (17 ml) at 4C for 15 min. Sections were pretreated for IHC assays after fixation by rinsing for 5 min in 0.05 mM Tris buffer, pH 7.6, containing 0.5 M NaCl and for 5 min in distilled water. Then sections were incubated in Lugol's solution for 5 min, which was followed by two rinses in distilled water, 5 min each, in 2.5% Na2S2O3 for 3 min, in tapwater for 5 min, and finally twice in rinsing buffer that contained 0.05 M Tris, pH 7.6, 0.5 M NaCl, and 0.5% Tween-20. To determine optimal dilution of primary antibody, we used concentrations of 10, 1, 0.1, and 0.01 µg/ml. For comparative studies of the various detection systems, we consistently used a concentration of 10 µg/ml of rabbit anti-rat cathepsin B (dilution 1:100; Upstate Biotechnology, Lake Placid, NY). Control incubations were performed by omitting the primary antibody or by replacing the rabbit anti-cathepsin B antibody by an irrelevant antibody, rabbit anti-salmon vitellogenin (BN-5; Biosense, Bergen, Norway) in the same dilution.
NanogoldSilver Staining Method.
The Nanogoldsilver staining method was performed according to
AvidinBiotin Complex with Diaminobenzidine (ABCDAB). Blocking of endogenous biotin was performed with avidin (Dako; Glostrup, Denmark) for 20 min at 20C. Sections were rinsed three times for 5 min in rinsing buffer. Blocking of endogenous avidin was performed with biotin (Dako) for 20 min at 20C and sections were rinsed three times for 5 min in rinsing buffer. Blocking of endogenous peroxidase was performed in 3% H2O2 in distilled water for 20 min at 20C. Sections were rinsed three times in rinsing buffer for 5 min at 20C. Blocking of nonspecific binding was performed in TMGS buffer at 20C for 20 min.
Incubation with rabbit anti-rat cathepsin B was performed in TMGS in buffer at 4C overnight. After incubation, sections were rinsed three times for 5 min in rinsing buffer. Then sections were incubated with the secondary biotinylated goat anti-rabbit antibody (Dako) diluted 1:300 in TMGS buffer for 30 min at 20C. Sections were rinsed three times in rinsing buffer for 5 min at 20C. Incubation with avidinbiotin complex (ABC system; Dako) was performed in a solution that was made up by adding 40 µl of streptavidin in PBS and 40 µl of biotin conjugated to horseradish peroxidase in PBS to 5 ml diluent for 30 min at 20C. Sections were rinsed in rinsing buffer for 5 min at 20C. Then sections were incubated in a solution of DAB and H2O2 (Dako) for 5 min at 20C. Sections were rinsed three times in distilled water for 5 min at 20C. Counterstaining was performed in Gill's hematoxylin for 1 min before mounting in Euparal.
Catalyzed Signal Amplification with DAB (CSADAB).
Sections were processed exactly as described for the ABCDAB method up to the rinsing steps after incubation with the ABC complex. Then sections were incubated with biotinylated tyramide (Dako) for 15 min at 20C. Sections were rinsed three times in rinsing buffer for 5 min at 20C. Sections were incubated in streptavidin-peroxidase conjugate (Dako) for 60 min at 20C and rinsed three times in rinsing buffer for 5 min at 20C. Then sections were incubated with DAB and H2O2, rinsed, counterstained, and mounted as described in the ABCDAB method. In some cases, 6 mg/ml ammonium-nickel sulfate was added to the DABH2O2 solution as recommended by
Catalyzed Signal Amplification (CSA) with NanogoldSilver Staining. Sections were processed exactly as described in the CSADAB method up to the rinsing steps after the incubation with biotinylated tyramide. Then sections were incubated with streptavidin conjugated with 1.4-nm Nanogold diluted 1:60 in TMGS buffer for 60 min at 20C. Then sections were treated as described in the Nanogoldsilver staining method from the rinsing steps after incubations with the secondary goat anti-rabbit antibody.
Microphotographs of polymerized DAB and counterstaining were made with an Axioskop microscope (Zeiss; Oberkochen, Germany) and either a x10 objective or a x40 water-immersion objective. Microphotographs of Nanogold-silver were made with the same microscope using epipolarized light.
Areas in sections positive for Nanogoldsilver were determined with the use of image analysis. A 3-chip CCD color video camera (Sony; ATV Horn, Aalen, Germany) was attached to the microscope and coupled via a frame grabber (maximal size 786 x 512 pixels) to an image analysis system with a KS 300 software package (Kontron; Eching, Germany). The camera signal and set-up were adjusted according to the recommendations of
Enzyme Histochemistry
The incubation medium to demonstrate cathepsin B activity consisted of 100 mM phosphate buffer (pH 6.0), 1.3 mM EDTA (disodium salt), 1 mM dithiothreitol, 2.67 mM L-cysteine, 1 mM 2-hydroxy-5-nitrobenzaldehyde (nitrosalicylaldehyde; Merck, Darmstadt, Germany) as coupling agent, and 1 mg/ml N-CBZ-Ala-Arg-Arg-4-methoxy-2-naphthylamide (Enzyme Systems Products; Livermore, CA) as substrate for cathepsin B (
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Results |
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Serial sections were used to compare sensitivity and specificity of the immunohistochemical and enzyme histochemical methods to detect protein and activity of cathepsin B in rat liver containing colon cancer metastasis. Activity was localized as yellow fluorescent granules against a green fluorescent background after an incubation period of 15 min at RT (Fig 2). Activity in liver parenchyma was distributed heterogeneously. Parenchymal cells around central veins (pericentral areas) contained lower activity than the remainder of the lobuli in all animals tested (Fig 2a and Fig 2c). The reaction was considered to be specific because control reactions in the presence of the selective cathepsin B inhibitor Z-Phe-Arg-FMK completely inhibited the reaction (Fig 2b). Tumors contained hardly any activity except for necrotic areas, where a rather high activity was found, and stroma that contained some activity (Fig 2a and Fig 2c).
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Immunolocalization with a 1:100 dilution of the primary anti-rat cathepsin B antibody demonstrated that the traditional Nanogoldsilver staining (Fig 3a) and ABCDAB methods with or without Ni-enhancement yielded hardly any positivity (Table 1). When CSA was included in the reaction, the sensitivity increased up to 10-fold (Table 1). Positivity was found in liver parenchyma, tumors, and particularly in necrotic areas (Fig 3b and Fig 3c). The reaction was specific because sections incubated with the irrelevant primary antibody against salmon vitellogenin were virtually unstained (not shown). Only a small amount of polymerized DAB was found in necrotic areas after control incubation. Ni-enhancement made the brown DAB precipitate black but it did not increase sensitivity (Fig 3b and Fig 3c; Table 1). However, the combination of CSA and Nanogoldsilver staining greatly increased sensitivity, whereas control reactions did not yield more product (Fig 3d3f; Table 1). In fact, 10 µg/ml primary antibody produced far too much signal for exact localization of cathepsin B protein (Fig 4a). Parenchyma was homogeneously and strongly positive, whereas tumors showed distinct protein labeling, particularly in stroma and necrotic areas. Tenfold dilution of the primary antibody (1 µg/ml) improved localization considerably (Fig 4b; Table 2). We found that a concentration of 1 µg/ml of the primary antibody gave the best results that were in agreement with localization patterns of activity of cathepsin B (cf. Fig 2 and Fig 4b). Higher dilutions of the primary antibody (concentrations of 0.1 µg/ml and 0.01 µg/ml) resulted in too little signal to show cathepsin B protein in sites where cathepsin B activity was present (Fig 4c). Again, the control reaction in the absence of primary antibody produced low amounts of staining (Fig 4d; Table 2). Similar results were obtained when the primary antibody was replaced by the irrelevant antibody.
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Reproducibility of amplification with CSA and Nanogoldsilver staining was very high (Table 3), provided that each step in the procedure was carefully controlled and standardized. In particular, the silver enhancement step appeared to be highly variable with temperature, period of incubation, and pH.
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When 1 µg/ml of the primary antibody and the combination of CSA and Nanogoldsilver staining were used, the following localization pattern of cathepsin B protein in rat liver containing colon cancer metastasis was found. Liver parenchyma contained homogeneously distributed high levels of cathepsin B protein, whereas the tumors contained low levels that were particularly localized in necrotic areas and stroma (Fig 4b).
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Discussion |
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The present study clearly indicates that combination of two amplification systems, i.e., the tyramide-dependent CSA method and the Nanogoldsilver staining method as introduced by Hacker and co-workers in ISH (
It must be stressed that every step in the staining procedure has to be carefully controlled to obtain low background levels and reproducibility. This is inherent to the enormous amplification of signal in the method, thus also of nonspecific signals (
Localization of cathepsin B protein and activity was largely similar in rat livers containing colon cancer metastases except for pericentral areas. Cathepsin B protein was homogeneously localized in liver parenchyma, whereas activity was distinctly lower in pericentral areas than in periportal areas. These lower levels of cathepsin B activity in pericentral areas were the only sign of cathepsin B in inactive form, be it as (pre)proform or bound to endogenous inhibitor(s) (
In conclusion, signal amplification with the combined use of CSA and Nanogoldsilver staining is a specific and highly sensitive technique for immunohistochemistry as it is for in situ hybridization (
Received for publication November 15, 1999; accepted March 2, 2000.
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