Proper Application of Antibodies for Immunohistochemical Detection: Antibody

Transcription

M I N I R E V I E W
Proper Application of Antibodies for
Immunohistochemical Detection: Antibody
Crimes and How to Prevent Them
Richard Ivell, Katja Teerds, and Gloria E. Hoffman
Leibniz Institute for Farm Animal Biology (R.I.), 18196 Dummerstorf, Germany; School of Molecular and
Biomedical Science (R.I.), University of Adelaide, SA5005, Australia; Department of Animal Sciences
(K.T.), Wageningen University, 6709 WD Wageningen, The Netherlands; and Department of Biology
(G.E.H.), Morgan State University, Baltimore, Maryland 21251
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For several decades antibodies raised against specific proteins, peptides, or peptide epitopes have
proven to be versatile and very powerful tools to demonstrate molecular identity in cells and
tissues. New techniques of immunohistochemistry and immunofluorescence have improved both
the optical resolution of such protein identification as well as its sensitivity, particularly through
the use of amplification methodology. However, this improved sensitivity has also increased the
risks of false-positive and false-negative staining and thereby raised the necessity for proper and
adequate controls. In this review, the authors draw on many years of experience to illuminate many
of the more common errors and problematic issues in immunohistochemistry, and how these may
be avoided. A key factor in all of this is that techniques need to be properly documented and
especially antibodies and procedures must be adequately described. Antibodies are a valuable and
shared resource within the scientific community; it is essential therefore that mistakes involving
antibodies and their controls are not perpetuated through inadequate reporting in the literature.
(Endocrinology 155: 676 – 687, 2014)
ntibodies, particularly for use in immunohistochemistry, represent one of the most powerful tools in
modern biological science. They combine extremely high
precision of identification at the protein level, with high
sensitivity, and also localization at a cellular or even a
subcellular scale. Although the technique of immunocytochemistry has been around for some 50 years (1), the
methodology itself is still relatively crude, and our understanding of what factors influence specificity and sensitivity is often rudimentary. In the postgenomic era of the
Internet we are inundated by information from companies
offering large numbers of antibodies, mostly against peptides or recombinant proteins, all of which postulate very
high specificity combined with rigorous controls. But how
much of this should we believe, and what are the minimal
controls that still need to be carried out to ensure adequate
scientific rigor in our experiments? Referees and journal
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ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2014 by the Endocrine Society
Received October 22, 2013. Accepted December 16, 2013.
First Published Online January 15, 2014
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endo.endojournals.org
editors are becoming alarmed by the often superficial way
in which antibody specificity is dealt with (2– 4). This
guide briefly discusses how antibodies are produced, how
they function in the context of immunohistochemistry,
and what controls and documentation are essential if a
result is to be believed. Unfortunately, the scientific literature is pervaded by examples of erroneous results using
antibodies, particularly in immunohistochemistry. Here
we hope to alert the investigator and potential referees to
the possible pitfalls that can be encountered.
What Are Antibodies? What Types Are
There? How Are They Generated?
This article cannot hope to summarize the vast amount of
very detailed literature concerning antibodies, their genAbbreviations: GST, glutathione-S-transferase; KLH, keyhole limpet hemocyanin.
Endocrinology, March 2014, 155(3):676 – 687
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serum may be further purified crudely, for example, using
the IgG-specific binding properties of Protein A or Protein
G, or by differential ammonium sulfate precipitation. Alternatively, an antiserum can be purified quite specifically
by using affinity chromatography binding to the original
immunizing antigen. Such affinity-purified antisera, although highly enriched in the specific IgG, may have lost
concentration (titer) because the highest affinity antibodies do not elute well from the columns, and/or may be
structurally damaged by the exposure to the very acidic pH
needed to elute the antibodies from the affinity column. In
general, species are chosen for immunization, which are
evolutionarily distant from either the species of the immunizing antigen, and/or the species in which the antibodies are to be applied. Most commonly, polyclonal antibodies are raised in rabbits, guinea pigs, donkeys, goats,
or sheep, although other species (eg, rats or chicken) may
also be used.
Monoclonal antibodies are created in much the same
way as polyclonal antibodies by the immunization of living mice (or sometimes rats). However, once a sufficient
titer of the specific polyclonal antibody is attained, the
animals are killed, their spleens are removed, and the individual antibody-producing lymphocytes (each producing a different individual but specific IgG) from the spleen
are immortalized by fusion with tumor cells, to produce
clonal cell lines (hybridomas) capable of making each specific IgG. Such clones are cultured in vitro to secrete into
the culture medium only one (monoclonal) antibody that
will recognize only a single immunogenic epitope.
Whereas most hybridoma cells are used to make IgG in
vitro by secretion into culture media, in the past, monoclonal antibodies could also be produced in vivo as ascites
fluid often with advantageously high titers, although this
procedure is ethically problematic (10).
In addition to IgGs, other Ig types may be generated.
For example, the mucosal immune system of the gut or
uterine lining generates preferentially IgA molecules in response to a mucosal surface immunogen. There are also
IgD, IgE, and IgM types of antibody al though these are not
relevant in the context of immunohistochemistry. Chicken
have proven very useful, because they can make IgY molecules in response to immunization, which are transported
to the egg yolk in large amounts, such that specific antibodies can be produced simply by collecting eggs, with
more than 1 mg of pure IgY in every egg (11).
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eration, and their functionality. There are numerous excellent reviews and books covering these topics (eg, Refs.
5–9). Here we intend to introduce only sufficient knowledge of antibodies to explain the issues that contribute to
the way they work in our experimental systems and, consequently, also their limitations and potential artifacts. In
general, antibodies are produced by B cells (or plasma
cells) within the body as part of the humoral response to
infection. Antibodies circulate in the blood or in peritoneal
fluid, or may be attached to the surface of lymphocytes,
and serve to interact specifically with foreign antigens,
causing these to be ingested by phagocytosis.
Antibodies are of several types, the most common being
IgG, which possesses 2 larger “heavy” chains, each linked
to shorter chains by disulfide bridges (Figure 1). The ends
of the chains form a hypervariable paratope (Figure 1),
which can specifically recognize a small 3-dimensional differentially charged surface (the immunological epitope) of
its cognate immunogen (the protein used for immunization), which we refer to as the “antigen” (meaning “antibody-generating” molecule). Upon infection or immunization, specific IgG molecules, and the cells producing
them, are clonally selected, and variability can be amplified by recombination and site-specific mutation within
these cells. Immunization of a living mammal with a large
immunogen gives rise to so-called polyclonal antisera, because many different IgGs are generated, each recognizing
a different 3-dimensional epitope within the same immunizing protein. For comprehensive details of immunization procedures, see Harlow and Lane (6, 7). Antisera are
the serum or sometimes plasma fractions from the blood
of immunized animals. The IgG fraction within the anti-
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Figure 1. Diagrammatic representation of an IgG molecule to indicate
terms used in the text. Fab fragments are those generated by papain
cleavage and comprise only a single antigen-combining site, whereas
F(ab)2 fragments are generated by pepsin cleavage and have 2 antigencombining sites.
Properties of Different Antibodies and
Immunogens
Individual monoclonal antibodies, or single IgG molecules, or the variable moieties of these, the so-called Fab
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Table 1.
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greater stability in a shorter time than those with low affinity and are therefore preferred in immunohistochemistry (12).
In between these two extremes are many of the antibodies that we use today, because they can be generated
quickly from just a DNA sequence. Such antibodies make
use of a small peptide sequence generated by translation of
a DNA or RNA sequence, followed by chemical synthesis.
Usually such peptides are about 10 –14 amino acids long
and without much secondary structure. They often represent an unconstrained (flexible) region of a protein that
is the equivalent of a single antigenic epitope (often a
group of 4 – 6 amino acids with charged or hydrophobic
side chains). Because they represent only single immunogenic epitopes, these antibodies, although polyclonal in
terms of their production, are little different from monoclonal antibodies and are referred to as monotypic antibodies. Like many monoclonal antibodies, they often have
only modest binding affinity and cannot be rigorously
washed. A brief summary of the main kinds of antibody
and their various advantages and disadvantages is given in
Table 1.
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fragments (fragment antibody-binding; peptidase-cleaved
IgG variable regions; Figure 1), all bind to their single
immunogenic epitope with high specificity but often only
modest affinity (ca. 10⫺5 to 10⫺7 M). This means that they
need to be applied in a relatively concentrated form (cell
supernatant dilutions of the order of 1:1000 or 1:100, or
even less are usual here), and washing procedures cannot
be too stringent because the specific binding could be ruptured. Because of the opportunity for clone selection in the
generation of monoclonal antibodies, it is possible also to
obtain higher affinity antibodies, although these are less
common. Polyclonal antisera, on the other hand, bind as
polyvalent entities, usually recognizing multiple immunogenic epitopes on a single target protein. Antibody detection relies on secondary antibodies or similar molecular
bridges, which build molecular networks between different IgG molecules binding to the same target, resulting in
cooperativity and binding affinities that are much greater
(eg, 10⫺8 to 10⫺12 M). This, in turn, allows for antibodies
to be applied at higher dilutions (eg, 1:1000 to 1:100 000
or greater) and also permits much more stringent washing.
Antibodies with high affinity, as in general most polyclonal antibodies, bind larger amounts of antigen with a
or
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Attributes of Different Common Antibody Types
Production
Conventional
polyclonal
Production time short
and inexpensive.
Require larger pure
proteins as
immunogens.
Monotypic polyclonal
Production time short.
Uses small peptides as
immunogens; hence,
easy to start from DNA
sequence.
Production time long and
expensive, requiring
substantial technical
skill. Procedure allows
for selection of optimal
antibodies.
Disadvantages
Greater likelihood of crossreactivity, high background,
and immune mimicry. Batchto-batch variability.
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Monoclonal
Advantages
Recognize multiple epitopes on
one antigen and can thus
help amplify a signal from a
target protein with low
expression level. Comprises
heterogeneous mixture of
different IgGs; hence, higher
affinity through
cooperativity, and thus more
resistant to rigorous
washing. More tolerant of
antigen variation; preferred
choice for detection of
denatured proteins. More
stable over a broad pH and
salt concentration.
Simple production procedure.
Can tailor antibodies to
specific peptide epitopes.
or
Antibody type
A hybridoma is a constant,
renewable source of identical
antibodies; hence, increased
reproducibility. Procedure
allows for selection for
optimal characteristics. Single
specific IgG means lower
background.
Single epitope recognition means
often lower affinity, and high
background and crossreactivity, also due to haptenrecognizing IgGs.
Often low concentration and
modest affinity; hence, less
rigorous washing possible.
High epitope specificity means
less tolerance of antigen
variation or damage; thus
highly susceptible to changes
in pH and salt concentration.
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Sometimes it is convenient to use a recombinant protein
made in Escherichia coli or in a eukaryotic cell line as
immunogen. These can produce very good antibodies, but
again there are aspects that require consideration. First,
such proteins may contain additional peptide sequences
used for their purification (eg, GST; glutathione-S-transferase), which may not subsequently be completely removed and may contribute to the polyclonal pool of IgG.
Second, as with the peptide sequences above, such recombinant proteins are often denatured because of the way
they are purified, eg, using 8 M urea, and consequently the
immunogenic epitopes may only be presented by denatured proteins and not by proteins in their natural in vivo
conformation. This may not matter if tissue sections are
automatically denatured, eg, by using an antigen-retrieval
procedure (13). But in many fixed or native preparations,
the natural protein may simply not exhibit any of the
epitopes against which the antibodies are directed. Occasionally, we have also observed that antibodies may be
present in such preparations that recognize contaminating
E. coli proteins, giving rise to a nonspecific signal, which,
however, can be effectively blocked by additionally using
a crude preparation of E. coli protein to preadsorb the
antibodies.
A clever variant of these immunogen methodologies,
which have been used to obtain a good effect in the localization of neurotransmitters in the brain, is to take account
of the postfixation chemistry by using reagents such as
formaldehyde, carbodiimide, or glutaraldehyde, in the
coupling of the immunogen to its hapten. In this way
the antibodies generated are able to recognize precisely the
molecular epitope present in the brain tissue that results
from fixation if the same agent is used (14, 15). Small
neurotransmitters, like many small molecules, are notoriously difficult to localize in tissue sections, because they
are quickly washed away unless there is rapid fixation or
conversion to a more easily complexed product.
Adjuvants (eg, Freund’s adjuvant) are complex molecular mixtures of known high immunogenicity (ie, they
provoke a strong immune reaction, for example, because
they contain pertussis toxin), which are added to the immunogen in order to stimulate the B cells and ensure the
production of antibodies. Although in themselves they
generally do not interfere with the use of antibodies for
purposes such as immunohistochemistry, it is important to
appreciate that all such antisera will also contain many
IgG molecules specific for the adjuvant or similar entities.
A more worrying aspect consequent to the boosting of the
animal’s immune system arises if the animal had been previously exposed to a different antigen. One striking case of
this was observed when the first specific antibody titer had
fallen to insignificantly low levels, and a different antigen
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It must be appreciated that short peptide sequences
alone are not strongly antigenic. The way such antipeptide
antibodies are made is to covalently link the small peptides
to a larger hapten protein (or other molecule), which itself
may be immunogenic, such as keyhole limpet hemocyanin
(KLH), BSA, or human thyroglobulin. Many hundreds of
identical small peptides covalently linked by either their
N- or C-termini can bind to one KLH molecule. In special
examples, more than one peptide from the same DNA/
RNA sequence (or protein) can be used, thus effectively
recreating a true polyclonal situation, but this is not common. What needs to be remembered for all such antibodies, is that they are mostly monotypic (having relatively
low affinity) and also that many IgG molecules are present
in the resulting antiserum that recognize the hapten molecule (eg, KLH). These are deliberately chosen for this
purpose, because haptens act as a kind of adjuvant, inducing a greater immune response. Where the antigen is
very small, for example, for some neurotransmitters or
amino acids, the chemistry by which the antigen molecule
is attached to the hapten becomes important and may need
to be taken into account during tissue fixation for immunohistochemistry (see Figure 2).
Figure 2. Importance of fixation for small molecule immunohistochemistry. A and B, To generate specific antibodies, the immunogen
was linked to a larger protein by carbodiimide. Only when carbodiimide is
used with the fixative (4% formaldehyde) (panel B), will the antigen be
presented to the antibody in the “right” form. C, Serotonin is not fixed
well with strong complexing fixatives such as acrolein (compare panel
D, where the mild fixative 4% paraformaldehyde was used). In
contrast, the small peptide TRH is only fixed by rapid strong fixation,
here using acrolein (cf. panels E and F).
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was injected followed by Freund’s adjuvant; this new serum contained significant amounts of both specific antibodies (16).
Finally, in this section, the increasing manufacture of
antibodies by so-called genetic immunization needs mention. In this procedure, rats or rabbits are immunized by
either sc or im injection, or by biolistic delivery (“genegun”), of a DNA expression construct which for some
days at least will be transcribed and translated in vivo to
yield a novel immunogenic protein, against which antibodies are generated (17). This method does not involve
the use of adjuvants or haptens, although sequences may
be incorporated into the DNA construct having a comparable effect, and most importantly, there is a very high
likelihood that the immunogen is presented in its native
conformation, and not denatured as is typical of most
recombinant proteins. We used this approach successfully
to produce antibodies recognizing a fetal antigen, against
which we had failed to develop antibodies by more conventional procedures (17).
response to immunogens previously, although may in fact
have moderate antibody titers against specific proteins in
their blood or could generate these (see above). Nor is it
acceptable to replace the primary antiserum simply with
PBS solution. If preimmune serum is not available, replacement of the primary antibody by an IgG of the same
class as the primary antibody may be an option. In this way
one can determine whether the primary antibody IgG
sticks to the section.
Validating Antibodies
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At the outset it needs to be made clear that there is no single
or perfect way to validate the specificity of antibodies used
for immunohistochemistry. Multiple approaches need to
be taken and judged by the “weight of evidence.” The
following is a critical appraisal of the many methods that
have been used to support the specificity of antibodies.
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Preadsorption of antibodies using the immunizing
antigen or equivalent
Here, usually prior to the application of the primary
antibody solution to a tissue section, the solution is preincubated with the immunizing antigen (peptide or protein, although usually not covalently linked to a hapten) in
considerable molar excess in order to quench any available
specific binding sites of the IgG for the target protein in the
section. The high molar excess is required usually because
of the relatively low affinity that particularly monotypic
antibodies have for their target, which will subsequently
be in equilibrium with the immunogen during the usually
long (eg, overnight) primary antibody incubation. Although
mostly a convincing control for antibody specificity, this
control will not exclude the possibility that antibodies are
recognizing a so-called immune-mimetic epitope in the
target tissue. This is a 3-dimensional molecular structure,
which, although not of identical sequence to the immunogen, has a similar molecular “shape,” which would be
equally recognized by the antibodies. Moreover, the
higher the concentration of antibodies used (see below),
the more likely that some immune-mimetic sequences may
be detected. It must be recognized, however, that if not
applied, the possibility of false-positive results greatly increases. It is better first to show that staining is blocked and
then to investigate the possibility for immune-mimicry.
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Omission of the primary antiserum
One of the most common controls used in immunohistochemistry is simply to leave out the primary antibody
and otherwise to complete the protocol identically to sections in which the primary antibody is included. Although
informative when high background staining is noted along
with more specific staining, this is probably the least useful
control for specificity, because it cannot distinguish any
false-positive (see later) results. It is a control for possible
nonspecific binding of the secondary antibody and says
little about the specificity of the primary antibody. If this
control is used, it is important to substitute the primary
antibody by an equivalent amount of a preimmune serum,
ideally from the same animal as was subsequently immunized, or failing that, at least normal serum from the same
species. Nonetheless, this will not establish specificity of
the generated staining: some other approach must be used.
The reason for using a preimmune serum from the same
animal is that for the sake of economy some antibodyproducing companies reuse animals for antibody production, which may not have generated a good immune
Western blotting
Western blots are frequently used (particularly in company catalogues) to prove the specificity of antibodies.
Here it is important to note, that it is essential to show the
whole molecular size range of the blot to exclude any unspecific binding to proteins which from their size could not
be the specific antigen. If there is a trail of nonspecifically
interacting proteins, then these will also bind antibodies in
tissue sections. It is not appropriate to perform Western
blots with a single purified recombinant protein, because
with only mild washing any Coomassie stainable band (ie,
in sufficient quantity) could bind nonspecifically and at
low affinity, suggesting a specific immune reaction. For
the same reason, it is important also to indicate just where
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cluded during the blotting procedure. Ironically, when controls for
Western blots are used, preadsorption with purified antigens is usually applied.
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Immunocytochemistry of
transfected cells
One of the simplest and clearest
of controls is to carry out transient
transfections of cell lines using various expression constructs and
then to apply cytospin immunocytochemistry for the transfected antigen (20) (Figure 3). The advantage of using transient transfection
is that there are both expressing
and nonexpressing cells for comparison within the same field of
view. In Figure 3 we, in fact, used 3
different expression constructs,
Figure 3. Cytospin preparations of human embryonic kidney293T cells transfected with
one for the target protein, a G
different expression plasmids encoding the human G protein-coupled receptors RXFP1, RXFP2, or
protein-coupled receptor called
HE6 (as indicated), and then subjected to immunocytochemistry using the double-peroxidase
RXFP1, and two for other more or
anti-peroxidase technique and polyclonal antibodies raised against the peptide antigen L7–2
(amino acids 278 –292 of the human RXFP1 sequence), coupled to KLH. In panels A and B
less closely related G protein-couantibodies derived from 2 independent rabbits immunized with the same L7–2 antigen were
pled receptors (all immunocytoused. Note in these panels using transiently transfected cells that there are both labeled (dark
chemically negative). Note also
stain) and unlabeled (pale cell outlines) cells. Panels C and D represent negative controls with
cells transfected with plasmids for non-cognate receptors. See Ivell et al (20) for more details.
that here we have used immune sera
from quite different rabbits (thus
having a different background pool
Coomassie-stainable proteins are running on Western
of polyclonal antibodies in addition to the specific moblots, because coincidence of the correct molecular size is
notypic antibodies raised against the common immunonot evidence of molecular identity, and any protein if in
gen). This technique can also easily be adapted to quanhigh enough concentration on a Western blot can bind
antibodies, albeit with only modest affinity. Good con- titative flow cytometry, although there is a caveat here
trols using Western blots are those using, for example, in that many commercial and other antisera contain
extracts of cells transfected or not with appropriate gene sodium azide as a stabilizer; this substance is toxic to
constructs; or possibly a panel of cells or tissues with cells even at low concentration, and hence would kill
known positive and negative expression profile, yielding living cells treated with such antibodies.
known specifically sized immunopositive bands, and no
others. Even this may not be sufficient, as a recent article
has shown (18), where protein sequencing was subsequently carried out to show that a band of the correct size
on a Western blot was not in fact the expected band, but an
immune-mimetic artifact. An excellent example of how antibodies can be evaluated using a Western blotting approach
is described by Panjwani et al (19). It is worth noting that it
is not always possible to obtain reliable Western blot results
using all antibodies: some antibodies demand a native conformation of the target protein not present on electrophoresed and blotted proteins; some molecules are too small for
Western blotting; and sometimes antigenic epitopes are oc-
Immunohistochemistry of tissues from genedeleted animals
An ideal control, where feasible, is to make use of tissues in which the target gene product of interest has been
genetically deleted. This is obviously only relevant for
those species, such as mice, in which an appropriate knockout animal is available, or where there is a natural mutation.
Because all other aspects of the tissue sections are identical,
this indeed offers a perfect control situation. The use of
knockout tissues is also ideal for validation of Western
blot approaches (19), especially to ensure that erroneous
secondary bands are not generated.
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monoclonal antibodies recognizing different epitopes
within the same protein immunogen can be very effectively
used in such a context. Here it is important to thoroughly
check that antibodies do indeed recognize discrete peptide
epitopes. In various company catalogues the same antisera
may be marketed under quite different catalogue numbers.
Also, it pays to check the peptide sequences given in such
catalogues, that when cross-reactivity is suggested for several species, a BLAST search of the GenBank database will
quickly show whether the antigenic peptide sequence is
indeed present in the species sequence of interest.
A further very convincing variant of this approach is to
make use of a quite different technique, such as in situ
mRNA hybridization (Figure 5) or laser capture RT-PCR,
to verify the cellular localization of the target protein of
interest and its absence in other cell types. Also such techniques can effectively target different
regions within a transcript (22), offering the added advantage of checking whether or not the signal represents a functional full-length
transcript. A variant of this approach, most easily carried out in
mice, is to take advantage of transgenically introduced unique proteins, such as ß-galactosidase, green
fluorescent protein, or Cre-recombinase, the tissue-specific expression of
which is driven by a promoter from
an endogenously expressed gene of interest (23–25). Sometimes, one can
also take advantage of the older techniques of enzyme-histochemistry (for
the localization of specific substratemetabolizing enzymes), or ligand
binding, where a specific protein-protein interaction (such as hormone-receptor binding) can be demonstrated
in a tissue section by hybridization
with radioactively or fluorescently labeled ligands (26 –28).
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Parallel reproduction of results using alternative
antibodies and/or techniques
Another very good control situation is to make use of
2 or more different antibodies recognizing different
epitopes within the same target protein, and which thus
should show coincidence of the cell type-specific signal.
An example is shown in Figure 4, in which 3 different
antibodies (L7–1, L7–2, and L7–3), each recognizing a
different peptide sequence within the human RXFP1 receptor, indicate that the same cell types within the endometrium are being recognized (cf Figure 4, A, D, and G).
Although ideally, one should use serial sections for such
analyses, it is possible to use nonserial sections, as here, if
the cell types are clearly identifiable, and the tissues used
are structurally and hormonally equivalent and have been
prepared and treated identically (21). Similarly, panels of
Figure 4. Sections (8 ␮m) of formaldehyde-fixed and paraffin-embedded endometrial tissue
from a nonpregnant macaque monkey of reproductive age, stained immunohistochemically
using the double-peroxidase anti-peroxidase technique and 3 different polyclonal antibodies
raised against 3 different peptide epitopes within the human RXFP1 protein sequence (panels
A–C, L7–1: amino acids 145–159; panels D–F. L7–2: amino acids 278 –292; panels G–I. L7–3:
amino acids 609 – 624). Using the immune sera (panels A, D, and G), significant cytoplasmic
staining is observed in endometrial stromal cells. However, there is also staining in the nuclei of
epithelial cells (arrowheads) using all 3 antibodies. All staining was eliminated when the primary
antisera were replaced by the corresponding preimmune sera from the same rabbits (panels B, E,
and H). However, only the epithelial nuclear staining disappeared when antibodies were
preincubated with an excess of KLH (panels C, F, and I), showing that this quasi-specific staining
was due to the presence of antibodies raised against the hapten KLH, which had been used in
the generation of all 3 primary antisera. For more details; see Ivell et al (20).
Antibody Titration, Signal
Amplification, and Effects
on Specificity
As already discussed, the ideal primary antibody for immunocytochemistry is one binding its specific
antigen with very high affinity. This
means that it can be applied at high
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molecules binding the specific antigen in a section should
only depend on the number of target molecules in the tissue and not on the antibody dilution. In practice, however,
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dilution, such that any nonspecific interactions, which are
generally low-affinity interactions, are obviated. Theoretically, for long incubation times, the number of antibody
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Figure 5. Double labeling of mRNA and protein in the same cell. A, GnRH. The image on the left shows GnRH mRNA using a biotinylated
riboprobe stained with immunoperoxidase detection of the biotin in black. B, Inversion converts the black product to white, and staining using
immunofluorescence in red shows that the same cell that possessed mRNA also possesses the product of that mRNA, GnRH peptide. C, Tyrosine
hydroxylase. As in panels A and B, but the cell in this image has its mRNA stained black and the protein tyrosine hydroxylase in brown.
Figure 6. In conventional heterologous immunohistochemistry (panel A), there is little risk that the secondary antibody will cross-react with
endogenous IgG in the tissue sections (here mouse-on-rat). Thus a biotin-peroxidase approach using a biotinylated second antibody (panel B) will
give rise to good specific staining (panel E). However, for homologous immunohistochemistry (here mouse-on-mouse; panel C), such secondary
antibodies will also react with the endogenous IgG in the tissue, giving rise to high unspecific background staining (panel F). One way that this
problem can be overcome is to use a primary antibody that has been purified and directly biotinylated (panel D), combined with a heterologous
goat-antibiotin antibody as secondary antibody. This would then be detected using a conventional conjugated antigoat tertiary antibody, to give
rise to an image (panel G) little different from the heterologous situation (cf. panel E). Ab, antibody.
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indirect immunohistochemical detection methods are used. This background staining is caused, in general,
by binding of the secondary antimouse antibody to endogenous
mouse tissue IgGs (Figure 6, C and F;
in this case omission of the primary
antibody is an appropriate and necessary control). Different approaches have been suggested to resolve this problem. In Figure 6, an
example is illustrated whereby the
primary mouse IgG has been purified
and directly biotinylated, allowing
the use of a secondary goat antibiotin
antibody, now giving comparable
staining specificity (Figure 6, D and
G) to the equivalent heterologous situation (Figure 6, B and E). Another
solution for this problem is to use
monovalent IgG Fab fragments that
recognize both the Fc and F(ab)2
regions of IgG as a preincubation
step (Figure 7). This step does not
affect the antigen specificity of the
primary antibody. Instead of the
normally used biotin-labeled goatantimouse secondary antibody, a biotin-conjugated F(ab)2 goat-antimouse antibody is then applied to the
tissue (Figure 7C and inset). This
leads to almost complete elimination
of
the
background staining (cf Figure 7A) (32).
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Figure 7. Immunohistochemical labeling of mouse testis tissue with a mouse monoclonal
antibody against the protein p62 (brown staining), a marker for autophagy. Examples of labeled
spermatogonia and primary spermatocytes are indicated by arrowheads. A, Conventional
immunohistochemical staining procedure, using normal goat serum and a biotinylated goatantimouse secondary antibody. Note that except for the cytoplasm of the elongating spermatids
all cells seem to stain. B, Omission of the primary p62 antibody, otherwise using the same
procedure as in panel A; the staining in spermatogonia and spermatocytes has more or less
disappeared, although background staining in most cells remains present. C, Normal goat serum
has been replaced by a preincubation with goat-antimouse Fab fragments, and the biotinylated
secondary goat-antimouse antibody has been replaced by a F(ab)2-labeled biotinylated goatantimouse antibody; now there is specific brown staining in the spermatogonia and primary
spermatocytes only and an absence of any background staining. The inset in panel C shows a
detail of this. D, As negative control, the p62 antibody has been replaced by IgG of the same
class as the primary antibody; otherwise the procedure is exactly as in panel C. Scale bars: panel
A, 42 ␮m; panel D, 10.5 ␮m.
where incubation times are finite, very high dilution may
also lead to signal attenuation. A way to overcome this is
to make use of antibody amplification systems, such as the
avidin-biotin-peroxidase complex method (29), the double-peroxidase anti-peroxidase/avidin-biotin-peroxidase
complex method (30), or more recently tyramine amplification (reviewed in detail by Hoffman et al in Reference 31). Signal amplification allows a higher dilution
of the primary antibody (and hence higher specificity),
although it should be remembered, that amplification
systems could also amplify mimicry with equal efficacy
if this is not eliminated.
Homologous Immunohistochemistry
Often it is necessary to use mouse monoclonal antibodies
on mouse tissues (Figure 6A), a procedure that is inevitably
complicated by high levels of background staining when
Specific Issues Relating to
Immunofluorescence
The use of fluorescently tagged antibody systems, especially combined with high-resolution microscopy, such as
laser confocal scanning microscopy, represents a revolution in morphological imaging. The great advantage of
using such fluorescence-based systems is that they capture
very narrow depths of field, and filters can be applied that
eliminate signals from other wavelengths, making detection highly selective for relatively narrow wavelength
ranges (ie, specific antibody signals). Data from multiple
wavelength ranges can then be overlaid in the computer to
assess colocalization. However, exactly the same problems regarding specificity and controls apply for
immunofluorescence as for conventional immunohisto-
03/06/2014
doi: 10.1210/en.2013-1971
685
calization and ranges of intensity within each structure.
Biologically, it would be hard to imagine 2 different proteins to be always present in exactly the same amounts in
exactly the same cells. Placement of a single-labeled specimen under both sets of filters controls for this “bleedthrough” phenomenon (33). Often if the weaker of the 2
antigens is used with the green fluorophore in doublelabeling with red and green, the problem can be avoided.
False Negatives and False Positives
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Figure 8. Relative intensity and separation of fluorophores. Lowsensitivity methods for immunofluorescence detection using a Cy2tagged secondary antibody usually only gives a signal in the green
channel. However, when such signals are amplified, for example using
a tyramine-based procedure, not only are strong signals evident in the
green emission channel (panel A), there is often sufficient “bleedthrough” fluorescence in the tail of the emission range of the green
fluorophore now to give a signal that is detected also as red (panel B,
here using very specific Texas Red filters), and the image appears as if
it is double-labeled, even though in this case only a single, Cy2-tagged
antibody has been used. A further control for this is to reverse the
order of the red and green fluorophores, using the weaker antigen as
green, and the stronger one as red. Note the highly suspicious
morphologic identity of the 2 images.
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A false negative is where, on the basis of other information, one would expect to see an immunohistochemical
signal, but this is not evident. Clearly, the antibodies themselves may not be specific or have the appropriate titer. But
sometimes a false-negative situation may occur where the
antigenic sequence of the target protein is in some way
hidden from the antibody or presented in a different way
because of fixation chemistry. One solution may be to use
an antigen or epitope retrieval procedure that applies a
strong chemical denaturation step or heat that exposes
subcellular and cellular structures not otherwise accessible
in conventional sections. For example, this approach has
worked well for transcription factors and nuclear receptors located within the nuclei of cells. One caveat to this
approach is that it will only work where the immunogenic
epitope is equivalently denatured, ie, for small peptide
immunogens, or for predenatured recombinant proteins.
An alternative source of false-negative results is when the
target antigen is relatively small and becomes washed out
from thin sections during processing. A simple solution
here can be to use different fixation chemistry, or possibly
to employ thicker sections (despite loss of resolution).
Just as different fixation procedures can give rise to
differing results, so also can antibodies behave differently
applied to fixed and paraffin-embedded tissues, compared
with frozen sections, or to cell culture preparations. Although the latter are like frozen sections in some ways,
generally such cells require specialist permeabilization
protocols (eg, using mild nonionic detergents, or saponin),
which allow for entry of an antibody into the cell (for
intracellular target antigens), without loss of cellular
structural integrity. Insufficient or excessive permeabilization or inadequate subsequent washing steps can easily
lead to false-positive or false-negative results, or even to
false subcellular localization (34). Otherwise, the kinds of
controls required are similar to those for conventional
tissue sections.
A false positive occurs, where, despite apparently good
and reproducible validation (see above), an antibody-specific signal is observed in cells in which no such signal is
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chemistry. Additionally, however, immunofluorescence is
accompanied by some problems of its own, the most common of which relates to quality and choice of suitable
fluorophores, and corresponding excitation and emission
filters (Figure 8). It is essential to control individually for
each fluorophore being used, to ensure that the chosen
wavelengths for the excitation and emission filters are indeed mutually discrete, especially when double labeling is
planned. Figure 8 illustrates a typical artifact where the tail
of emission from a bright green fluorophore falls within
the range of emission from a red fluorophore. This could
easily be confused with the specific signal coming from a
red fluorophore (note: in the example illustrated in Figure
8, this slide had only one fluorescent molecule that had
peak emission in the green range; no second fluorophore
was applied, and yet there is evidently a marked signal
corresponding to the red fluorophore). The most obvious
giveaway for such an artifact is that the immunofluorescence for what should be the second primary antibody
appears so similar to that for the first antigen that it is
simply “too good to be true” in terms of its cellular colo-
03/06/2014
Antibody Crimes
used are fully documented with proper identification (eg,
catalogue and preferably lot or batch numbers, indicating
bleed and/or animal identity). Newly created antibodies
require a full description of the immunizing antigen (as
now demanded in the current Instructions to Authors for
Endocrinology and other journals). A failure to provide
rigorous documentation on identity and specificity of antibodies represents grounds for rejecting scientific work.
Acknowledgments
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We thank the many students, colleagues, and authors who have
directly and indirectly taught us much about the quirks and trials
of using antibodies. We also want to acknowledge particularly
the contributions of Ms Marga Balvers (Hamburg) and Dr. Wei
Wei Le, a long-time colleague and friend of Dr. Gloria Hoffman,
who was instrumental in devising some of the strategies presented in this article. She died in June, 2013.
Address all correspondence and requests for reprints to:
Richard Ivell, PhD, Leibniz Institute for Farm Animal BiologyReproductive Biology, Wilhelm-Stahl-Allee 2, Dummerstorf,
Germany 18196. E-mail: [email protected] or richardivell@
gmail.com.
This work was supported by the following agencies: Deutsche
Forschungsgemeinschaft, National Health and Medical Research Council of Australia, Australian Research Council, and
US National Institutes of Health-National Institute of Neurological Disorders and Stroke.
Disclosure Summary: None of the authors has anything to
declare.
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expected, or where other techniques (eg, in situ mRNA
hybridization; immunohistochemistry of knockout animals) suggest that no signal should be present. The most
obvious cause is an impure antigen or the presence of an
immune-mimetic epitope (as described above). The former requires knowledge of the source of the immunogen;
the latter is common when the epitope is identical to or
conformationally similar to sequences in other unrelated
proteins. Controlling mimicry can be difficult and in this
regard can be minimized through use of polyclonal rather
than lower affinity monotypic or monoclonal antibodies¸
where stringent washing is often not feasible. A further
cause of false positives can be that antibodies are present
that have been generated against moieties in the immunogen unrelated to the target antigen. For example, we had
observed what looked like a very specific signal in cell
nuclei of the macaque uterine epithelium (Figure 4 and
Reference 20). We had used a peptide-specific antibody
generated using KLH as hapten. However, this signal occurred only in sections from the macaque monkey, and not
in human, and could be completely eliminated by preincubation of the antibodies with pure KLH (Figure 4, C, F,
and I). Similar examples are known in which antibodies
have been raised against recombinant proteins retaining a
GST moiety in the immunogen, and which then recognize
GST wherever this is expressed, eg, in the male reproductive tract. This may also occur when thyroglobulin or BSA
has been used in preparation of the immunogen. Such issues may be resolved by using a panel of monoclonal antibodies, if available, recognizing different epitopes within
the same target molecule, and which, because of selection,
cannot recognize the hapten.
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Ivell et al
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686
Conclusions
The list of controls and possible artifacts encountered in
immunohistochemistry summarized here cannot be exhaustive. Ultimately, it comes down to the “weight of evidence” in favor of specificity or against it. But in publishing immunohistochemical results it is essential that this
evidence is presented adequately, so that informed readers
can make up their own minds. What is equally essential is
that information on company websites is regarded cautiously and that companies begin to specify the methodology they have used for both staining and tissue preparation. In presentation of data using antibodies, authors
should be aware that a reference to someone else’s work
where a different mode of tissue acquisition and/or a different method of detection in a different location is used,
may not provide adequate evidence of control for their
experiment. Authors should ensure that the antibodies
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Proper Application of Antibodies for Immunohistochemical Detection: Antibody

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