Volume 46(8): 977–983, 1998
The Journal of Histochemistry & Cytochemistry
http://www.jhc.org
TECHNICAL NOTE
A New Blocking Method for Application of Murine Monoclonal
Antibody to Mouse Tissue Sections
Qi L. Lu and Terry A. Partridge
Muscle Cell Biology, Medical Research Council Clinical Science Centre, Hammersmith Hospital, London, United Kingdom
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 biotin–streptavidin immunohistochemical detection protocol with biotinylated anti-mouse F(ab9)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 46:977–983, 1998)
SUMMARY
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 (Guerin–Reverchon et
al. 1989). However, a limitation of indirect immunohistochemistry is that primary Abs raised in a given
Correspondence to: Q.L. Lu, Muscle Cell Biology, MRC Clinical Science Centre, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK.
Received for publication December 9, 1997; accepted April 8,
1998 (7T4545).
© The Histochemical Society, Inc.
0022-1554/98/$3.30
KEY WORDS
immunohistochemistry
monoclonal antibody
homologous tissue
background blocking
species usually cannot be applied to tissues from that
same species. This is exemplified by murine MAbs,
which are difficult to apply to the detection of antigens in mouse tissues by indirect methods, largely, it is
believed because of the presence of endogenous mouse
immunoglobulins (Igs) which are also recognized by
the secondary Ab, creating a nonspecific background.
The level of background is tissue type-related and varies from sample to sample. Within tissue sections the
homologous Igs are normally present in intercellular
spaces and therefore cause particular problems for the
detection of antigens localized at or near the cell surface or in the extracellular matrix, because the resulting background staining has the same distribution as
positive signals for specific primary Ab. Detection of
membrane antigens with homologous Ab becomes
even more difficult when the Ab works only on cryostat sections without fixation, because the persistence
of native protein is often associated with higher background compared to immunostaining on fixed-frozen
or paraffin sections. No blocking method has been
977
978
fully satisfactory (Nielsen et al. 1987; Yamashita and
Korach 1989; Hierck et al. 1994).
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 (Nicholson et al. 1989) and the mdx
mouse (Hoffman et al. 1990). To analyze the nature
of revertant dystrophin, we wished to immunostain
revertant fibers with a panel of MAbs, each recognizing specific exons (exon mapping). However, all the
exon-specific MAbs are of murine origin and work
well only on unfixed cryostat sections with indirect
immunohistochemical detection, conditions that predispose to strong intercellular background staining
which masks any membrane-localized specific signals
for dystrophin. The alternative of using directly labeled primary MAbs (Tse and Goldfarb 1988) is insufficiently sensitive and is wasteful of Abs that are in
short supply. We have therefore studied factors affecting background staining in mouse tissues with primary murine MAb detected by indirect immunoperoxidase or immunofluorescence methods. We confirm
that binding of secondary anti-mouse Ab to endogenous mouse tissue components is the main cause of
the background staining, and accordingly have evolved
a rationale for blocking this binding by preincubation
with Fab of papain-digested unlabeled secondary Abs
plus Fc fragment of the anti-mouse Igs. This blocking
technique in combination with use of the F(ab9)2
fragment of secondary Ab in place of whole labeled
secondary, led to the most complete elimination of
background staining. As a result, we were able to demonstrate low levels of dystrophin unequivocally in revertant fibers by both immunoperoxidase and immu
nofluorescence methods. This simple blocking system
can be adapted to the use of other Abs on homologous
tissues.
Materials and Methods
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
Lu, Partridge
from 3FTG b-galactosidase transgenic mice were also examined for nuclear b-galactosidase expression. Composite
blocks of muscle samples from C57BL/10 and mdx mouse
were used for initial tests. Sections 6 mm thick were cut onto
3-aminopropyltriethoxysilane (Sigma; Dorset, UK)-coated
slides, dried, and stored at 270C 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 b-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 HRPconjugated swine anti-rabbit (SAR) Igs (0.8 mg/ml, 1:200 dilution; DAKO).
For the three-step biotin–streptavidin 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(ab9)2 (1:200; DAKO)
was also used as secondary Ab. Enzyme activity of HRP was
developed with 3,39-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 al-
Monoclonal Antibody on Homologous Tissues
979
bumin (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 peroxidase–antiperoxidase (mPAP) immunostaining method, in which mAb
to dystrophin was used as primary Ab. The digested antimouse 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 DAB–
0.1% 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 12–16 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 220C for longer periods.
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 mg/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 0.1–0.5 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
Figure 1 The principle for use of the mPAP method to determine
the efficiency of papain digestion. (A) Normal mPAP immunohistochemistry using anti-mouse Igs as the bridge between the primary mouse Ab and the mPAP complex. Signal is produced specific
for the primary mouse Ab. (B) mPAP immunohistochemistry using
papain-digested anti-mouse Igs as second layer. Complete digestion with papain reduces the anti-mouse Igs to Fc and monomeric
Fab fragments, thus abolishing their ability to bridge the primary
mouse Ab and the mPAP complex. As a result, no signals for primary mouse Ab (or endogenous tissue Igs) are produced.
initial incubation time ranged from 15, 30, 45 min to 1 hr at
RT and 12 hr at 4C.
Results
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
980
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.
Muscle tissues are rich in endogenous biotin
(Kirkeby et al. 1993). The level of background staining was lower with the two-step peroxidase or fluorescence detection system than with the biotin–streptavidin method, suggesting that endogenous avidin–biotin
binding was involved in the high level of background.
When sections were blocked with the Biotin Blocking
System (DAKO) followed by the biotin–streptavidin
detection system, a reduced level of background staining was observed in most samples but, again, not
enough for a clear identification of dystrophin in revertant fibers with MAbs. Therefore, endogenous tissue biotin contributes to but is not the main cause of
background staining in these muscles.
We then investigated the blocking efficiency of papain-digested rabbit anti-mouse Fab initially using the
biotin–streptavidin method as a detection system. A
significant reduction in background staining was observed when Fab of RAM Igs was used at 0.05–1 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 im-
Lu, Partridge
possible 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 papaindigested 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
Figure 2 Three-step immunoperoxidase detection of dystrophin in mdx mouse muscle with MAb MANDYS8 and biotinylated RAM Igs as a
second layer. (A) Section without blocking. Two groups of fibers (*) with slightly stronger membrane staining than the rest of the fibers are
observed. The revertant nature of these fibers is difficult to establish because of moderate levels of interfiber background staining. (B) Section (adjacent to A) blocked with RAM Fab. The two groups of fibers can be unequivocally identified as revertants with greatly reduced
background. (C) Section (next to B) blocked with RAM Fab-c. The revertant fibers are demonstrated, with all background eliminated. Bar 5
80 mm.
Figure 3 Three-step immunofluorescence (streptavidin–Texas red) detection of dystrophin on adjacent sections of denervated mdx mouse
muscle with MAb MANDYS8. (A) Section visualized with biotinylated RAM Igs as a second layer without blocking. A strong background signal masks any possible signal specific for dystrophin. (B) Section blocked with RAM Fab-c and detected with biotinylated RAM Igs as a second layer. Fibers with stronger membrane signal than the remaining background are observed but the revertant nature of these fibers is
difficult to establish. (C) Section blocked with RAM Fab-c–Fc and detected with biotinylated RAM IgG F(ab9)2 as a second layer. The revertant
fibers are unequivocally demonstrated with background mainly restricted to the damaged fibers. Bar 5 50 mm.
Figure 4 Three-step immunofluorescence (streptavidin–Texas red) detection of b-galactosidase in 3FTG mouse muscle with MAb GAL13.
(A) Section without blocking and detected with biotinylated RAM Igs as a second layer. It is difficult to distinguish the nuclear signal for
b-galactosidase above the strong background. (B) Section blocked with RAM Fab-c and detected with biotinylated RAM IgG F(ab9)2 as a second layer. The nuclear signals are clearly demonstrated in the absence of background. Bar 5 80 mm.
Monoclonal Antibody on Homologous Tissues
Ab to mouse tissue components was further supported
by the result obtained using F(ab9)2 as the secondary
Ab. Muscle sections blocked with Fab-c/Fc and then
visualized with biotinylated RAM F(ab9)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 biotin–streptavidin 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 b-galactosidase
in peripherally localized muscle nuclei.
Figure 5 Immunofluorescence double labeling of dystrophin with MAb
MANDYS8 (recognizes exons 31–32)
detected with FITC-conjugated GAM
Igs (A) and polyclonal Ab p6 (recognizes exons 57–60) detected with
Texas red-conjugated DAR Igs (B). Two
groups of revertant fibers are differentially demonstrated: Both groups of
fibers are detected with p6, whereas
one of them is not detected with
MANDYS8, suggesting a diversity of
dystrophin protein in the revertant fibers. The section was blocked with
GAM Fab-c. Bar 5 80 mm.
981
The effective blocking with Fc fragments of antimouse 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 nonantigen-specific binding of Abs to mouse Fcg 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 recog-
982
nized 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 biotin–
streptavidin 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.
Discussion
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. Nielsen et al. (1987) reported that background staining could be eliminated
when human IgM was used as primary Ab on human
tissues by preincubation of sections with monomeric
Fab of unlabeled rabbit anti-human IgM and when the
same batch of labeled rabbit anti-human IgM was
used to detect the primary Ab. However, when the
primary Ab was human IgG, only limited reduction in
background was achieved by the same strategy. This
difference may be attributable to the low tissue concentration of IgM such that background staining is often negligible, even without blocking, when a classspecific secondary Ab is used. The limitation of Fab
blocking led to efforts to develop alternative methods
for application of Ab on homologous tissues (Lewis
Carl et al. 1993; Hierck et al. 1994). Hierck et al.
(1994) reported a single-step indirect detection method
in which mouse MAb and labeled anti-mouse secondary Ab were allowed to form complexes in solution
followed by blocking of the unoccupied paratopes of
the secondary Ab with Igs in normal mouse serum.
The whole reaction mixture was then applied to
mouse tissue sections. However, this method suffers
Lu, Partridge
from poor accessibility of the large Ab–Ab complexes
to tissue antigens and thus from low sensitivity. Furthermore, the ratio for primary and secondary Abs
must be optimized individually for each set of Abs,
making it more difficult to obtain consistent and comparable results when different batches of Ab are used.
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 (Nielsen et al. 1987) that failed to
achieve satisfactory blocking with Fab of the same
batch of unlabeled IgG, suggesting a possible binding
of the Ab through sites other than Fab. Binding of Fc
does not appear to be through the non-polymorphic
FcR II/III receptor of endogenous mouse Igs, because
blocking with Ab to the FcRs did not significantly reduce background. Interestingly, this binding cannot be
blocked by high levels of normal rabbit Igs or its papain digests but can be effectively inhibited by the Fc
fragment of anti-mouse Igs. This suggests that Fc of
anti-mouse Igs has higher binding affinity than Fc of
nonimmune serum of the same species, with the implication that immune rabbit serum contains a different
spectrum of Ig isotypes from nonimmune serum, with
Fc of greater nonspecific binding capacity to mouse
tissue components. There is no particular reason why
this should apply only to mouse tissues, and this phenomenon of nonspecific binding of Fc of immune secondary serum may underlie sporadic high background
staining encountered when the primary Ab is not of
the same species as the tissue being stained or when a
polyclonal primary Ab is used. It is clear that background staining in the system studied above is multifactorial, and only limited reduction in background is
achieved by blocking tissue sections for biotin binding
or with Fab individually. Complete blocking was
achieved in samples with moderate to heavy background only when sections were blocked against binding of both Fab and Fc of secondary Ab to tissue Igs
and other components. The use of a crude papain digest
of unlabeled secondary Ab for blocking provides a simple preparation that may provide Fab with strong antigen binding efficiency, which may possibly be attenuated
during purification procedures. However, the most
complete blocking was achieved by addition of extra
Fc to the crude papain digest. In some tissues, blocking the binding of endogenous biotin or peroxidase activity may also be required.
In summary, our study shows that background
staining encountered in the application of Ab to ho-
983
Monoclonal Antibody on Homologous Tissues
mologous 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 papaindigested secondary anti-mouse Igs enriched with the
Fc fragment of the same Igs. This, in combination with
the biotin–streptavidin 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.
Acknowledgments
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.
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