TECHNICAL NOTE |
Correspondence to: Qi L. Lu, Muscle Cell Biology, MRC Clinical Science Centre, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK.
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Summary |
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Antigen detection with primary antibody of the same species as the test tissue is complicated by high levels of background staining when indirect immunohistochemical detection methods are used. This severely limits the use of murine monoclonal antibodies on tissues of the mouse, the most widely used experimental model system; no method for blocking this is fully satisfactory. Here we show that background staining encountered in this system results largely from the binding of secondary antibodies via both Fc and Fab to endogenous immunoglobulins and other tissue components. A simple and efficient blocking strategy was established, employing papain-digested whole fragments of unlabeled secondary anti-mouse Igs enriched with Fc fragment of the same Igs. We have used this method to visualize dystrophin, an antigen expressed at low level, in revertant fibers of mdx mouse by both immunoperoxidase and immunofluorescence methods. In combination with the use of a biotinstreptavidin immunohistochemical detection protocol with biotinylated anti-mouse F(ab')2 as second layer, we eliminated the heavy background in this system and achieved strong signal amplification to demonstrate the specific antigen clearly. Double labeling with one mouse antibody and one antibody from another species was performed without signal interference. This principle can be adapted for wider applications, such as antibodies of other species on homologous tissues and perhaps where high background is found with heterologous antibodies. (J Histochem Cytochem 46:977983, 1998)
Key Words: immunohistochemistry, monoclonal antibody, homologous tissue, background blocking
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Introduction |
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Immunohistochemistry is the most powerful method for localizing a specific antigen to particular cell types in a heterogeneous population or to specific compartments within a cell. The sensitivity of the technique is much enhanced by amplification systems involving indirect detection of the primary antibody (Ab) by one or more additional steps. For example, the primary Ab can be detected either with secondary Ab labeled with an enzyme or fluorescent marker (two-step method) or by a biotin-labeled secondary Ab followed by avidin conjugated to enzyme or fluorescent labels (three-step method). The latter is the most powerful and permits the visualization of antigen expressed at very low levels (
We have encountered these problems in studies involving the detection of dystrophin in mouse muscles with murine MAbs. The dystrophin gene encodes 14-KB mRNA and 427-kD protein which can be detected at the subsarcolemma of normal skeletal muscles by indirect immunofluorescence or immunoperoxidase methods. However, it is too minor a component of the sarcolemma to be visualized reliably by directly labeled Ab. Dystrophin is lacking in muscles of boys with Duchenne muscular dystrophy (DMD) and in the mdx mouse homologue of this disease. Interestingly, sporadic individual muscle fibers that express dystrophin, called "revertant" fibers, are found in muscles of both DMD patients (
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Materials and Methods |
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Tissue Samples and Section Preparation
C57BL/10 and mdx mice aged from 3 months to 1 year were used. Muscles (five samples from five C57BL/10 mice and 25 samples from 20 mdx mice, including four muscle samples denervated 21 or 28 days previously) were dissected and snap-frozen in isopentane cooled by liquid nitrogen. Muscles from 3FTG ß-galactosidase transgenic mice were also examined for nuclear ß-galactosidase expression. Composite blocks of muscle samples from C57BL/10 and mdx mouse were used for initial tests. Sections 6 µm thick were cut onto 3-aminopropyltriethoxysilane (Sigma; Dorset, UK)-coated slides, dried, and stored at -70C until use.
Antibodies
MAb MANDYS8 (IgG2, 1:100 dilution; Sigma) and polyclonal rabbit Ab p6 (1:500 dilution, a kind gift from P. Strong, Neuromuscular Research Unit, Hammersmith Hospital, London, UK), both against dystrophin, were used for initial background and blocking tests on composite blocks of C57BL/10 and mdx mouse. A panel of six exon-specific MAbs to dystrophin, MANEX45A (IgG1, 1:4 dilution), MANEX50 (IgG1, 1:4 dilution), MANDYS101(IgG2b, 1:5 dilution), MANDYS110 (IgG1, 1:5 dilution), (all culture supernatants kindly supplied by G.E. Morris, MRIC Biochemistry Group, The North East Wales Institute, Wrexham, UK), NCL-DYS1 (IgG2b, 1:5 dilution; Novocastra, Newcastle Upon Tyne, UK), MANDRA1 (IgG1, 1:100 dilution; Sigma), as well as MAb GAL13 (IgG1, 1:100 dilution; Sigma) to ß-galactosidase, D33 (IgG1, 1:50 dilution; DAKO, Cambridge, UK) to desmin were examined.
Immunohistochemistry
Sections were air-dried, hydrated in PBS, and incubated for 30 min with nonimmune normal serum (1:20 dilution) of the animals of the species in which the second layer Ab was raised, and then blocked with 1% H2O2 in PBS for 20 min (for immunoperoxidase staining only), followed by primary Ab for 1 hr at room temperature (RT).
For two-step detection methods, primary mouse Abs were detected with FITC-conjugated rabbit anti-mouse (RAM) Igs (0.4 mg/ml, 1:40 dilution; DAKO) or goat anti-mouse (GAM) Igs (0.8 mg/ml, 1:50 dilution; DAKO) or with horseradish peroxidase (HRP)-conjugated RAM Igs (1.3 mg/ml, 1:100 dilution; DAKO). Primary rabbit Ab p6 was detected with Texas red-conjugated donkey anti-rabbit (DAR) Igs (0.9 mg/ml, 1:200 dilution; Amersham Lifescence, Poole, UK) or HRP-conjugated swine anti-rabbit (SAR) Igs (0.8 mg/ml, 1:200 dilution; DAKO).
For the three-step biotinstreptavidin detection method, sections were incubated with biotinylated RAM Igs (reacts with all mouse IgG subclasses, IgA and IgM, cross reactions with rat Igs and fetal calf serum have been removed; (0.6 mg/ml; 1:400 dilution; DAKO), or swine anti-rabbit (SAR) Igs (0.7 mg/ml, 1:500 dilution; DAKO) for 45 min followed by HRP- or Texas red-conjugated streptavidin (1:200 dilution; Amersham). Biotinylated RAM F(ab')2 (1:200; DAKO) was also used as secondary Ab. Enzyme activity of HRP was developed with 3,3'-diaminobenzidine tetrahydrochloride (DAB, 1 mg/ml PBS; Sigma) and 0.1% H2O2 for 4 min and the sections were counterstained with hematoxylin.
For double immunofluorescence staining, one of the MAbs and the polyclonal rabbit Ab p6 were used. Sections were incubated with both Abs simultaneously for 1 hr, followed by FITC-conjugated GAM Igs (1:50 dilution; DAKO) and Texas red-conjugated DAR Igs (1:200 dilution; Amersham) for 1 hr. All Abs were diluted in 3% bovine serum albumin (BSA; Sigma) in PBS and intervening washes of sections were carried out twice with PBS for 5 min unless otherwise stated. Negative controls were carried out by replacing the primary Ab with 3% BSA in PBS or by MAb NCL-DYS3 (IgG, 1:5 dilution; Novocastra), which is specific for human dystrophin and does not crossreact with mouse dystrophin.
Papain Digestion and Fab and Fc Preparations
Unlabeled RAM Igs (purified Ig fraction and reacts with all mouse IgG subclasses, IgA and IgM; 3.2 mg/ml; DAKO), RAM IgGs (purified IgG and reacts with mouse IgG, IgA, and IgM; 2.1 mg/ml; Sigma) and goat anti-mouse Igs (reacts with mouse IgG, IgA, and IgM; 1.6 mg/ml; DAKO) were incubated with papain (5% of Igs, w/w; Sigma), 20 mM L-cysteine (Sigma), and 1 mM EDTA, pH 7.4, for 4, 8, 12, and 16 hr at 37C. Digestion was terminated by addition of iodoacetic acid to a final concentration of 10 mM (Sigma) for 30 min at RT and was assessed by the mouse peroxidaseanti-peroxidase (mPAP) immunostaining method, in which mAb to dystrophin was used as primary Ab. The digested anti-mouse Igs was used (2 mg/ml) as a bridging second layer in place of undigested whole anti-mouse Igs followed by mPAP (1:50 dilution; DAKO) for 1 hr and developed with DAB0.1% H2O2 for 4 min. As illustrated in Figure 1, complete digestion, which would eliminate bivalent binding of Ab, was signaled by the absence of any staining in comparison to the presence of staining obtained when the undigested whole anti-mouse Igs was used. Optimal digestion was achieved after 1216 hr of incubation. The crude papain digests (referred to as Fab-c) were stored as aliquots at 4C for use within 1 month or at -20C for longer periods.
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Fab and Fc fragments were separated from papain-digested Igs or IgGs by affinity chromatography using a HiTrip protein G affinity column (Pharmacia Biotech; Herts, UK) according to the manufacturer's instructions. Blocking efficiency of Fab or Fc was compared only with Fab-c from which the Fab and Fc were prepared.
Background Blocking
The following methods were examined for blocking efficiency: (a) washing with 1% Triton X-100 (Sigma) in PBS for 1 hr at RT before application of normal serum incubation; (b) incubation with fragments of papain-digested unlabeled anti-mouse Igs before application of primary Ab (see below); (c) blocking the binding of avidin to endogenous tissue biotin using a Biotin Blocking System (DAKO) according to the manufacturer's instructions; (d) blocking with mouse Fc receptor (FcR) blocking Ab, rat IgG2 anti-mouse CD16/CD32 MAb (Pharmingen; Cambridge, UK) at 10 µg/ml for 1 hr before application of primary Ab; and (e) Incubation with purified normal rabbit Igs (DAKO) at a concentration of 2 mg/ml or with a papain digest of these Igs in place of normal rabbit serum. Papain-digested anti-mouse Igs and IgGs were used for blocking in the following forms and combinations: (a) Fab-c at a concentration ranging from 0.1 to 1 mg/ml; (b) Fab at a concentration of 0.10.5 mg/ml; (c) Fc at a concentration of 0.1 mg/ml; (d) Fab and Fc at the concentrations of 0.2 and 0.1 mg/ml respectively; and (e) Fab-c and Fc at the concentrations of 0.2 mg/ml and 0.1 mg/ml respectively. The initial incubation time ranged from 15, 30, 45 min to 1 hr at RT and 12 hr at 4C.
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Results |
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Dystrophin was localized with rabbit anti-dystrophin Ab p6 at the sarcolemma of all muscle fibers in normal C57BL/10 mice. However, in the mdx mouse only a few sporadic fibers were positively stained (revertant fibers), all remaining fibers being negative for dystrophin when two- or three-step detection methods were used. When MAbs were used without blocking, background staining ranging from mild (five samples), to moderate (16 samples) to heavy (nine samples, including four denervated muscles) was observed. In sections with moderate background, a slightly stronger than background staining was suggestive of revertant fibers but could not be used as a reliable indicator (Figure 2A), whereas in sections showing heavy background revertant fibers were not distinguishable (Figure 3A). Therefore, revertant fibers could not be identified in most samples of mdx mouse with MAbs. Revertant fibers could be identified in samples with mild background only when the MAb-stained sections were viewed together with an adjacent section stained with polyclonal Ab p6. Background staining at similar intensity remained even when MAb NCL-DYS3 (specific to human dystrophin) was used or the primary Abs were omitted. That background was caused by the secondary anti-mouse Abs was supported by the fact that no background staining was observed when primary rabbit anti-dystrophin Ab was followed by biotinylated SAR Igs and peroxidase-conjugated streptavidin. A limited reduction in background was achieved when sections were prewashed with 1% Triton X-100 for 1 hr at RT with constant shaking. This reduction, however, was not sufficient for clear demonstration of revertant fibers in muscles with moderate and heavy background. Triton X-100 washes did not reduce specific staining for dystrophin, because signal intensity remained unchanged when polyclonal Ab p6 was applied to such washed sections.
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Muscle tissues are rich in endogenous biotin (
We then investigated the blocking efficiency of papain-digested rabbit anti-mouse Fab initially using the biotinstreptavidin method as a detection system. A significant reduction in background staining was observed when Fab of RAM Igs was used at 0.051 mg/ml with an optimal incubation time of 1 hr at RT (this duration was used thereafter unless otherwise stated). As a result, positive staining for dystrophin in revertant fibers was clearly identifiable with mAbs in 10 muscle samples, although weak background persisted (Figure 2B). These muscles showed intrinsically low to moderate background in the absence of blocking with Fab. However, in samples with innately heavy background, such as denervated muscles, it remained impossible to clearly identify revertant fibers, and Fab concentrations higher than 0.2 mg/ml did not reduce background staining further; rather, it augmented it. Failure to completely block the background could be due to the difference in binding specificity between secondary Ab and blocking Igs from which Fab was prepared. However, this proved not to be the case because a similar result was obtained when the blocking Fab was prepared from the same batch of RAM Igs that had been biotinylated as the secondary Ab.
Background staining, however, was almost eliminated in all muscle samples that showed low to moderate background (21 of 30 samples) when papain-digested Fab-c, containing both Fab and Fc of RAM Igs or IgGs, was used at concentrations between 0.1 and 0.5 mg/ml, being optimal at 0.2 mg/ml. This is shown in Figure 2C, where dystrophin in revertant fibers was unequivocally demonstrated with no background staining. In addition, this blocking treatment, when applied to muscles with heavy background, revealed some revertant fibers, although visible background persisted (Figure 3B). This result suggested that Fc of the RAM Igs was involved in the elevated background staining, possibly due to binding to endogenous mouse tissue Fc receptors or other tissue components. Because Fab-c at a concentration higher than 0.2 mg/ml was associated with increasing background, we used the Fc fragment alone or together with Fab-c for blocking. Fc of RAM IgGs alone reduced background staining to a greater extent than Fab alone, to a level similar to that with Fab-c in most muscle samples. When Fc (0.1 mg/ml) was used together with Fab-c (0.2 mg/ml) for blocking on sections with heavy intrinsic background, further reduction of background was achieved, below the level that could be attained with Fab-c blocking alone.
The conclusion that background staining was partly caused by binding of Fc fragments of secondary Ab to mouse tissue components was further supported by the result obtained using F(ab')2 as the secondary Ab. Muscle sections blocked with Fab-c/Fc and then visualized with biotinylated RAM F(ab')2 followed by Texas red or HRP-conjugated streptavidin showed a complete elimination of background in moderate background specimens. Even in the intrinsically high-background denervated muscles, background was barely visible with this regimen (Figure 3C) and, in combination with the three-step biotinstreptavidin amplification, allowed clear identification of staining for dystrophin in revertant fibers in all mdx muscle samples.
An unequivocal identification of nuclear antigen in muscle fibers was also possible with this system. This is shown in Figure 4 for detection of ß-galactosidase in peripherally localized muscle nuclei.
The effective blocking with Fc fragments of anti-mouse Igs raised the possibility that a similar blocking effect could be obtained by incubation with high concentrations of normal rabbit Igs. This, however, proved not to be the case. Sections incubated with normal rabbit serum at 1:1 dilution, or with purified nonimmune rabbit Igs at up to 2 mg/ml or its crude papain digests, produced no noticeable reduction of background. To see whether mouse Ig FcRs were responsible for the binding of the Fc fragment of the secondary anti-mouse Abs, sections were incubated with rat anti-mouse CD16/CD32 MAb, which blocks non-antigen-specific binding of Abs to mouse Fc II/III receptor. Reduction in background was barely noticeable, suggesting that Fc of the secondary Ab binds to mouse tissue components other than the FcRs recognized by the Ab. When blocking with Fab-c/Fc at a concentration of 0.2 mg/0.1 mg/ml was applied to the two-step detection system with HRP- or FITC-labeled RAM Igs, specific signal for dystrophin and desmin, as expected, was slightly weaker than with the biotinstreptavidin detection system, with background negligible in all muscle samples.
To examine whether blocking with Fab-c was also applicable to double fluorescent staining, sections were blocked with Fab-c of GAM Igs at a concentration of 0.2 mg/ml and then incubated with one MAb to dystrophin or desmin and polyclonal rabbit Ab p6 to dystrophin followed by FITC-conjugated GAM Igs and Texas red-conjugated DAR Igs. Signals specific for each Ab were clearly demonstrated without interference between them. As shown in Figure 5, double staining with exon-specific Abs P6 and MANDYS8 identified revertant fibers expressing dystrophin with variation in exon composition on the same section. Whereas dystrophin expressed in almost all revertant fibers was stained by P6 to the C-terminal region, exon 31 to 32 detected by MANDYS8 was lost in a large proportion of revertant fibers.
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Discussion |
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The background staining found when murine MAbs were applied to mouse tissues and detected by indirect methods was considered to be largely due to the presence of tissue Igs homologous with the primary Ab. If this were so, it should be possible to block the binding of secondary Ab to the tissue Igs by prior occupation of the binding sites with an Fab monomer derived from unlabeled secondary Ab.
Our present investigation showed that failure to develop a satisfactory blocking method is due to the fact that background staining is not caused by a single factor when a homologous Ab is used. We found that endogenous peroxidase activity and the presence of endogenous tissue biotin and Igs all played some part but, surprisingly, that one major effect was the binding of the secondary Ab through its Fc fragment to tissue components. This was, in fact, consistent with the previous result (
In summary, our study shows that background staining encountered in the application of Ab to homologous tissues and detected by indirect immunohistochemistry mainly results from the binding of both Fab and Fc of secondary Abs to tissue Igs and other components. A simple and efficient blocking strategy was established that employs the unpurified papain-digested secondary anti-mouse Igs enriched with the Fc fragment of the same Igs. This, in combination with the biotinstreptavidin immunohistochemical detection system, effectively eliminates heavy background caused by the secondary Ab while providing maximal signal amplification, thus allowing detection of antigens expressed at low level with homologous Abs. Double labeling can be achieved with the blocking without signal interference. The principle and system can be adapted for wider applications in which Abs of other species can be used on homologous tissues and perhaps, in some instances, when unexplained high background staining is found with Ab and tissues of different species.
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Acknowledgments |
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Supported by an MRC program grant and the Leopold Muller Bequest.
We are indebted to Professor Glenn Morris (MRIC Biochemistry Group, The North East Wales Institute, Wrexham, UK) and Dr Peter Strong (Neuromuscular Research Unit, Hammersmith Hospital) for Abs used in this work and to Drs Jennifer Morgan and Christian Pastoret, who kindly provided tissues from mdx and c57 mice.
Received for publication December 9, 1997; accepted April 8, 1998.
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