TECHNICAL NOTE |
Correspondence to: Chris M. van der Loos, Academical Medical Centre, Dept. of Cardiovascular Pathology (H0-120), Meibergdreef 9, NL-1105 AZ Amsterdam, The Netherlands. E-mail: c.m.vanderloos@amc.uva.nl
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Summary |
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The newly developed Animal Research Kit (ARK) offers a simple and economic way of biotinylating mouse primary antibodies for background-free immunostaining of mouse and rat tissue specimens. Biotinylation involves the use of a biotinylated goat anti-mouse immunoglobulin Fab fragment mixed with a mouse primary antibody and subsequent blocking with normal mouse immunoglobulin. Because a reliable immunoenzyme double staining procedure on human tissue specimens with two unlabeled mouse primary antibodies of identical subclass is almost impossible, we have tested the performance of ARK biotinylation of one primary antibody in a multistep indirect/direct staining protocol. The multistep double staining procedure involved the subsequent application of an unlabeled mouse monoclonal antibody (MAb) 1 detected with an enzyme-labeled EnVision reagent, normal mouse serum for blocking, followed by a biotinylated mouse MAb 2 and enzyme-labeled streptavidin. Alkaline phosphatase and peroxidase enzymatic activities were developed last. Double staining results obtained with an ARK biotinylated reagent were compared with a truly biotinylated reagent using N-hydroxy succinimidebiotin for conjugation. It appeared that both biotinylation procedures revealed identical double staining results. Although a limited number of antibody combinations have been tested, it is clear that this double staining procedure will be successful for many antibody pairs. (J Histochem Cytochem 48:14311437, 2000)
Key Words: human tissue, immunohistochemistry, double staining, EnVision, biotinylation, Fab fragment, Animal Research Kit, identical IgG subclass
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Introduction |
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IMMUNOENZYME DOUBLE STAINING has become a valuable investigatory tool frequently applied in many biological sciences. For many applications, the long-lasting character of colored reaction products has been considered an advantage over high staining accuracy but rapidly fading fluorochrome signals. Before starting an immunoenzymatical double staining procedure, many considerations and selections should be made. Most important is the choice of a proper double staining concept (
The Animal Research Kit (ARK) was originally designed for background-free immunohistochemical staining with mouse primary antibodies on tissue specimens from any species, including mouse and rat. The procedure is based on in vitro labeling of a mouse primary antibody with a biotinylated anti-mouse Fab fragment, resulting in biotinylation of the primary antibody. Remaining biotinylated anti-mouse Fab fragments are subsequently blocked with normal mouse immunoglobulins (
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In this study we have compared on serial sections the use of an ARKbiotinylated reagent with a "true" N-hydroxy succinimidebiotin conjugate. After observing no difference between the ARK biotin reagent and "true" biotin conjugate, six straightforward mouse primary antibody combinations with identical IgG subclass were tested on cryostat and paraffin-embedded tissue specimens, including the application of heat-induced antigen retrieval (
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Materials and Methods |
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Tissue Preparation
The following human tissue specimens were used for cryostat sections: two tonsils with nonspecific changes obtained at surgery from young children and two aortic segments with atherosclerotic lesions obtained at autopsy. Tissue samples were snap-frozen in liquid nitrogen and stored at -80C. Six-µm cryostat serial sections were cut and dried overnight under a fan. Tissue sections were numbered and stored dry at -80C. On use, the sections were fixed in pure acetone (10 min, 4C) and briefly air-dried. Endogenous peroxidase activity was blocked with 0.1% sodium azide + 0.3% peroxide in 50 mM Tris-HCl-buffered saline, pH 7.8 (TBS; 20 min, room temperature) (
Reagents
Primary antibody pairs used for double staining are listed in Table 1. EnVision/goat anti-mouse IgG, HRP (EnVision/GAMHRP), EnVision/goat anti-mouse+rabbit IgG, AP (EnVision/GAM+GARAP), normal mouse serum, normal goat serum, AP-conjugated streptavidin, HRP-conjugated streptavidin, and ARK kit (consisting of a biotinylation reagent and a blocking reagent; other kit components were not used in this study) were from DAKO (Glostrup, Denmark). D-Biotinyl--amidocaproic acid N-hydroxy succinimide (ANHSbiotin) ester was from Roche/Boehringer (Mannheim, Germany); 3-amino-9-ethylcarbazole (AEC), naphthol-AS-MX-phosphate, Fast Blue BB (cat. no. 3378), and levamizole were from Sigma (St Louis, MO).
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CD68, EBM11/biotin
The IgG1 fraction from 10 ml anti-CD68, EBM11 was purified using a Agaroseprotein A column and buffers from Pierce (Rockford, IL), according to the instructions of the manufacturer. The yield was 1.3 mg of purified anti-CD68, EBM11 in 1.5 ml Tris-HCl (50 mM, pH 7.8) stored at -80C in 500-µl aliquots. One aliquot (= 430 µg) was biotinylated with ANHSbiotin ester according to the instructions from the manufacturer (Roche/Boehringer), using a molar ratio of biotin ester and immunoglobulin 10:1. After biotinylation, the conjugate was tested immunohistochemically by applying a 60-min incubation at room temperature. AP-conjugated streptavidin was used as second step, and Fast Blue BB as chromogen. The optimal dilution of the CD68, EBM11/biotin conjugate was found near 1:200.
In Vitro Biotinylation with ARK
The DAKO ARKulator (cat. no. S3953), a software application based on Microsoft Excel, was used for calculation of the reagent volumes needed for in vitro biotinylation. The regular working dilution of the antibody (equal to the dilution used for a regular three-step streptavidin detection technique), specific mouse immunoglobulin concentration of the antibody, and desired working volume were input data, and the volumes of primary antibody, buffer, biotinylation reagent and blocking reagent were read-outs. The in vitro biotinylation of the second mouse MAb with biotinylated goat anti-mouse Fab fragments was done according to the instructions with the ARK reagents. For example, for the anti-CD68, EBM11 antibody, 2.5 µl of antibody (395 µg/ml specific IgG) was first diluted in 468 µl TBS, and 10 µl biotinylation reagent (goat anti-mouse Fab/biotin) was added. After incubation for 15 min, 20 µl of blocking reagent (normal mouse immunoglobulin) was added to block the unbound biotinylation reagent (5 min). The reagent was now ready for use. Input data of the other primary antibodies were based on the dilution and IgG concentration as presented in Table 1.
Double Staining Procedure
Antisera and antibodyenzyme conjugates were diluted in TBS + 1% bovine serum albumin (BSA). TBS washings were performed between all steps (three times for 2 min), and all incubations were performed at room temperature unless otherwise stated. Normal goat serum was applied first for blocking nonspecific binding. The multistep double staining technique was performed as previously described (
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After application of all the immunoreagent steps, first AP activity was developed in blue (515 min, RT) using Tris-HCl buffer (100 mM, pH 8.5) and including naphthol-AS-MX-phosphate as substrate (0.2 mg/ml) and 1 mM levamizole (0.12 mg/ml) as inhibitor of endogenous AP activity. Just before use, Fast Blue BB was added as chromogen (0.2 mg/ml). After rinsing the sections with TBS buffer, HRP activity was developed in red (515 min, RT) using AEC as chromogen (0.5 mg/ml) in sodium acetate buffer (50 mM, pH 5.2). Just before use, 0.01% perhydrol was added as substrate.
Controls consisted of replacing one of the primary antibodies with a nonimmune mouse antibody of identical subclass. Specific IgG concentration and Ig isotype/subclass were matched with the specific primary antibody from the original double staining experiment. For the CD3/CD25 combination, the detailed conditions of the matched IgG1 controls are given in Table 1. All primary antibodies in Table 1 were also used for single immunostaining, using the two-step EnVision/HRP technique. After the double staining experiments, a comparison was made with the original single staining slides.
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Results |
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The two multistep double staining experiments, according to the scheme in Fig 1 using either an ARKbiotinylated or an ANHSbiotinylated anti-CD68, EBM11 antibody performed on serial sections showed identical results with respect to both single and double stained structures (Fig 2 and Fig 3). Three additional antibody combinations on cryostat sections are shown in Fig 4 Fig 5 Fig 6: von Willebrand factor/E-selectin, CD4/CD8, and CD3/CD25. Fig 7 Fig 8 Fig 9 represent double staining experiments performed on paraffin sections after heat-induced antigen retrieval with citrate buffer: Ki67/cytokeratin, vimentin/HMB45, and CD68/-actin.
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All primary antibodies involved in this study displayed comparable staining patterns with either single or double immunoenzyme staining and matched the staining pattern descriptions provided by the supplier. In all performed control experiments, the replaced antibody revealed completely negative staining.
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Discussion |
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In this study we have demonstrated equal double staining results with either ARKbiotinylation or a true biotin conjugate, thus showing that the method of biotinylation did not affect the outcome. Therefore, the DAKO ARK, although designed for different purposes, can be used successfully for in vitro biotinylation of unlabeled anti-human mouse MAbs to be used in a multistep double staining protocol. The obvious advantage of ARKbiotinylation is the requirement of only minute amounts of a primary antibody compared with traditional ANHSbiotin conjugation. Eight tested antibody combinations show distinct basic colors and occasionally a mixed color at sites of co-localization. Unwanted crossreactions among the applied reagents were never observed.
An alternative double staining procedure with two antibodies of similar IgG subclass resembling the present technique, was described by
Nevertheless, some limitations of the present double staining procedure should be noted. First, the ARKbiotinylation is based on a calculation requiring a specific mouse Ig concentration. Consequently, only purified commercial antibody products with a known mouse immunoglobulin concentration, which should be stated in the data sheet, can be properly employed for in vitro ARKbiotinylation. Second, the observation of a mixed color at sites of co-localization is generally limited to two antigens that occur with almost equal staining intensities (
Although the eight tested double staining combinations in this study still are limited, one can assume that the ARKbiotinylation procedure, in combination with the multistep indirect/direct double staining technique, can be theoretically applied for an indefinite number of mouse monoclonal primary antibody pairs. This double staining protocol is completely independent of the mouse antibody Ig (sub)class and the availability of primary antibodies in a conjugate format. The general protocol outlined in Table 2 may serve as a "blueprint" for many successful double staining combinations.
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Acknowledgments |
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We wish to thank DAKO A/S (Glostrup, Denmark) for financial support for color print reproduction and Mr Mikkel Nielsen for supporting us with reagents and information related to the ARK. We are also indebted to Prof Dr Anton E. Becker for critical reading of our manuscript.
Received for publication November 11, 1999; accepted April 12, 2000.
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