D'Amico2008State of The Art in PDF

ARTICLE IN PRESS
JIM-10849; No of Pages 18
Journal of Immunological Methods xxx (2008) xxxxxx
Contents lists available at ScienceDirect
Journal of Immunological Methods

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j i m
Review
State of the art in antigen retrieval for immunohistochemistry

Fabio D'Amico , Evangelia Skarmoutsou, Franca Stivala
Department of Biomedical Sciences, University of Catania, Via Androne 83, 95124 Catania, Italy
a r t i c l e
i n f o
Article history:
Received 19 January 2008
Received in revised form 19 November 2008
Accepted 19 November 2008
Available online xxxx
Keywords:
Antigen retrieval
Immunohistochemistry
Fixation
a b s t r a c t
The masking effects of antigens by chemical xation, processing, embedding media
interactions, represent a serious problem for immunohistochemical purposes. Fortunately,
different approaches in antigen retrieval exist. These techniques are relatively recent and
continuously expanding.
This review focuses on the present state of the art in antigen retrieval methods for
immunohistochemistry in light and electron microscopy. Moreover, a brief discussion on the
chemical aspects of xation, mechanism of retrieval, as well as its efcacy, is given.
2008 Elsevier B.V. All rights reserved.
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . .
The mechanism of antigen retrieval . . . . . . . .
Retrieval efcacy. . . . . . . . . . . . . . . . . .
3.1.
Overview . . . . . . . . . . . . . . . . . .
3.2.
Fixation effect . . . . . . . . . . . . . . .
3.3.
Heat effect . . . . . . . . . . . . . . . . .
3.4.
pH effect . . . . . . . . . . . . . . . . . .
3.5.
Metal ions effect . . . . . . . . . . . . . .
3.6.
Solution type effect . . . . . . . . . . . . .
3.7.
Molarity effect . . . . . . . . . . . . . . .
3.8.
Non specic background . . . . . . . . . .
Light microscopy application . . . . . . . . . . . .
4.1.
Enzymatic digestion . . . . . . . . . . . . .
4.2.
Heat . . . . . . . . . . . . . . . . . . . .
4.3.
Heat and enzymatic digestion combination .
4.4.
Ultrasound . . . . . . . . . . . . . . . . .
4.5.
Minor approaches. . . . . . . . . . . . . .
4.6.
Approaches for non embedded specimens and
Electron microscopy application . . . . . . . . . .
5.1.
Some considerations for electron microscopy .
5.2.
Enzymatic digestion . . . . . . . . . . . .
5.3.
Heat . . . . . . . . . . . . . . . . . . . .
5.4.
Chemical bleaching and etching . . . . . . .
5.5.
Different approaches combinations . . . . .
5.6.
Minor approaches . . . . . . . . . . . . .
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Corresponding author. Tel./fax: +39 095312017.

E-mail address: f.damico@unict.it (F. D'Amico).
0022-1759/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jim.2008.11.007
Please cite this article as: D'Amico, F., et al., State of the art in antigen retrieval for immunohistochemistry, J. Immunol.
Methods (2008), doi:10.1016/j.jim.2008.11.007
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ARTICLE IN PRESS
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F. D'Amico et al. / Journal of Immunological Methods xxx (2008) xxxxxx
6.
A brief note on standardization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
7.
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
1. Introduction
Immunocytochemistry plays an important role in diagnostic histopathology. The sequential processes of xation,
dehydration and embedding in light and electron microscopy,
produce extensive molecule modications, often affecting
adversely protein antigenicity, which may result in some
cases in failure (Brandtzaeg, 1982; Puchtler and Meloan, 1985;
Larsson, 1988; Grifths, 1993). Aldehyde xation has been
recognized as one of the major causes for immunohistochemical failures in the past (Berod et al., 1981; Brandtzaeg, 1982).
In the last two decades, several methods were developed in
order to unmasking or retrieving the antigens for immunohistochemical purposes (Shi et al., 1991; Werner et al., 1996;
Gown, 2004).
Antigen retrieval techniques may signicantly increase the
sensitivity of the immunohistochemical detection of epitopes,
occasionally paralleling the sensitivity achieved in frozen/
unxed tissues, but with better morphologic detail and more
precise antigen localization (van den Berg et al., 1993;
Iwamura et al., 1994; Taylor et al., 1996b; Sheriffs et al., 2001).
Several acronyms, often synonymous and misleading, for
such techniques were generated in literature. Some of them
are listed in Table 1.
The antigen retrieval can be obtained by physical,
chemical approaches, or mixture of both (Table 2). The choice
of method is antigen and/or antibody dependent, reecting
the different, not fully dened and diverse mechanisms of
antigen retrieval technology (Shi et al., 1993; Boon and Kok,
1994; Xiao et al., 1996).
In general, the technical principle of antigen retrieving
could involve the breaking of protein cross-links, introduced
by xation process, with the subsequent exposition of antigen
sites to antibody.
Antigen retrieval offers other additional advantages. In
fact, with the lowering of the threshold for detection of
antigens, it allows the use of higher antibody dilutions,
Table 1
Acronyms for antigen retrieval found in literature
Acronyms used in literature
AR, AGR
HBAR
HIAR
HIER
HMAR
HTAR
LTHMAR
LTER
MAR
PIER
WBH-AR
Antigen retrieval
Heat-based antigen retrieval
Heat induced antigen retrieval
Heat induced epitope retrieval
Heat-mediated antigen retrieval
High-temperature antigen retrieval
Low temperature heat-mediated antigen retrieval
Low temperature antigen retrieval
Microwave antigen retrieval
Proteolytic induced epitope retrieval
Water-bath heating antigen retrieval
Most of them are synonyms.
which, other than economic advantage, lowers the possibility

of background staining, and increases the labelling specicity.
Moreover, antigen retrieval, minimizing the occurrence of
false negative results, raises the reproducibility of results and,
thus the diagnostic accuracy (Charalambous et al., 1993; Boon
and Kok, 1994; Shi et al., 1997; Leong and Sormunen, 1998).
Finally, whenever possible, antigen retrieval avoids the use
of frozen sections, which are more difcult to obtain with a
good morphology (Saito et al., 1992; McQuaid et al., 1995;
Taylor, 1996), and allow us the making of retrospective studies
on stored specimens.
Retrieval antigen technology is also very useful for other
techniques using histochemical labelling detection systems
such as ow cytometry, in situ hybridization, terminal deoxynucleotidyl transferase-mediated nick labelling (TUNEL), histochemistry of nucleic acids. These approaches are not reviewed
in the present paper. Interested readers are referred to Shi et al.
(2001), Yamashita (2007), for recent reviews. Furthermore,
antigen retrieval technology is also applied in proteomic studies
(Fowler et al., 2007).
2. The mechanism of antigen retrieval
Aldehydes represent the most commonly used xatives.
In particular, formaldehyde and glutaraldehyde are the
major xatives for light and electron microscopy purposes,
respectively.
The major effect introduced by aldehyde xation is the
cross-linking of protein amino acid residues by methylene
bridges (French and Edsall, 1945; Pearse, 1980; Fox et al., 1985).
Aldehyde xation may also be responsible for molecular
changes in carbohydrates, nucleic acids and phospholipids
(Baker, 1966; McGhee and von Hippel, 1977; Foster et al., 2006).
The degree of crosslinking depends on several factors, such as
the xation time and temperature, as well as xative pH and
concentration (Larsson, 1993).
The rst reaction between formaldehyde and a peptide
sequence causes the formation of a methylol (hydroxymethyl)
group on an amino acidic residue, such as arginine, cysteine,
histidine, lysine, tryptophan, tyrosine, serine, asparagine,
glutamine. Subsequently, Schiff's bases (imine groups) can be
formed by condensation of methylol groups on lysine residues.
These instable Schiff's bases condense with other groups, such
as phenolic, indole, imidazole groups of arginine, asparagine,
glutamine, histidine, tryptophan, cysteine and tyrosine, to make
methylene bridges (Fraenkel-Conrat and Mecham, 1949;
Kunkel et al., 1981; Shi et al., 2000; Vani et al., 2006; Metz
et al., 2004, 2006) (Fig. 1). Among the different amino acidic
residues, arginine, tyrosine and lysine residues are very
reactive, whereas asparagine, glutamine, and histidine residues
show a weaker reaction (Metz et al., 2006). Metz et al. (2004)
have found that methylene bridges were not formed between
two primary amino groups, but between a primary amino
group and other amino acid residues.
Methods (2008), doi:10.1016/j.jim.2008.11.007
ARTICLE IN PRESS
Table 2
Some examples of chemical and physical approaches used in antigen
retrieval
Chemical approach
Enzymatic digestion
Proteinase K, trypsin, chymotrypsin,

pronase, pepsin, N-glycanase F,
hyaluronidase
Denaturant and chaotropic
Formic acid, guanidine hydrochloride,
treatments
guanidine thiocyanate, urea, boric acid,
acetic acid SkipDewax, sodium dodecyl
sulfate, citraconic acid
Bleaching (oxidizing treatment) Periodic acid, hydrogen peroxide,
sodium meta periodate
Etching
Sodium (potassium) hydroxide
in (m)ethanol
Detergent treatment
Triton X-100
Physical approach
Heat treatment
Source: microwave, autoclave, pressure

cooker, steamer, water bath.
In solution of: distilled water, sucrose,
EDTA, EGTA, TBS, aluminum chloride, zinc
sulfate, lead thiocyanate, citrate buffer,
borate.
Ultrasound treatment
Such a schematic subdivision is not rigorous, because many approaches
result by the combination of two or more treatments. For example, most
chemical treatments are performed by heat. Furthermore, some substances
can show different chemical effects.
Glutaraldheyde shows a similar mechanism of action.

It reacts predominantly with the lysil residues, although
other reactions can occur with histidyl, tyrosyl and sulfhydryl
groups (Habeeb and Hiramoto, 1968).
Intra- and intermolecular links induced by aldehyde xation
alter the protein secondary, tertiary and quaternary structures
and lower the accessibility of epitope sites (Fraenkel-Conrat
et al., 1947; Fraenkel-Conrat and Olcott, 1948; Horobin, 1982;
Dapson, 1993). In contrast to this mechanism of denaturation,
Mason and O'Leary (1991) have stated that polypeptide chains
would preserve their secondary and tertiary structure. Similar
conclusions were made by Rait et al. (2004) whose spectroscopic studies showed that formaldehyde xation did not
perturb the secondary and tertiary structure of RNase A.
The mechanisms of antigen retrieval are not yet well
established and many theories exist. These include the breaking
of crosslinks, renaturation, formation of holes and gaps,
extraction of diffusible proteins, precipitation, stabilization,
and rehydratation of protein epitopes.
Although not completely, the antigenicity restoration is
mainly obtained by hydrolytic breaking of the intra- and
intermolecular crosslinks (Shi et al., 1991; Suurmeijer and
Boon, 1993a; Montero et al., 2000). Such a mechanism was
conrmed by recent studies using SDS-PAGE, which showed
that heat disrupts the crosslinks of proteins, induced by aldehyde
xation (Rait et al., 2004; Yamashita and Okada, 2005a).
Volkin and Klibanov (1989) have suggested that antigen
retrieval by heat would reverse the protein conformation,
altered by xation, inducing a renaturation of epitopes.
Emoto et al. (2005) hypothesized that, after disruption of
methylene crosslinks by heat, the antigen extends and its
hydrophobic and hydrophilic portions are exposed. At neutral
pH, the epitopes are hidden by an entangling process of the
protein, caused by the simultaneous presence of hydrophobic
and ionic forces. On the contrary, at acidic or basic pH, the

antigen molecules are now charged negatively or positively
and, the electrostatic repulsion and hydrophobic attractive
forces avoid cooperatively the occurrence of the entangling
process. However, at basic pH values, ionic strength can
reduce the electrostatic repulsion and, thus, the hydrophobic
forces cause the adverse entangling process.
As suggested by Morgan et al. (1994, 1997), calcium ions
are believed to be responsible, together with formaldehyde,
to form protein cage-like structures, which would reduce
the formation of antigenantibody reactions. According to
these authors, antigen retrieval by heat in citrate buffer or in
buffer containing EDTA or EGTA could remove such calcium
ions, thus permitting the unmasking effect. Because at low
pH values calcium chelation does not occur, acidic solution
would allow a calcium dissociation and a return to the
protein native state, whereas, basic solution would cause a
calcium chelation.
However, Shi et al. (1999), suggested that the exposure to
calcium ions results in conformational modication of some
epitopes, such as thrombospondin recognized by some
monoclonal antibodies, Ki-67 by MIB1 or p53 by 1810
monoclonal antibodies, but equally clearly others are unaffected. Moreover, they suggested that calcium-induced antigen binding inhibition is independent on formalin-induced
crosslinking.
Yamashita and Okada (2005a,b) demonstrated biochemically and immunohistochemically that EDTA did not show any
effect on the retrieval approaches for different antigens, such
as ER, ER, laminin, -amylase, glucocorticoid receptor, and
so on. Moreover, they excluded the presence of such cage-like
Fig. 1. Simplied scheme of formaldehyde reaction with proteins.

(a) Formaldehyde is added to a protein with the formation of a reactive
hydroxymethyl molecule (also called methylol group). (b) Formation of an
imine group (also called Schiff's base). (c) Methylene bridge formation
between a lysine residue (lysyl group) and nitrogen of a peptide linkage.
Formaldehyde is depicted as methylene glycol, formed by reaction with water.
Methods (2008), doi:10.1016/j.jim.2008.11.007
ARTICLE IN PRESS
4
structures with calcium ions and concluded that calcium

effects act for some antigens only.
In conclusion, the role of calcium in antigen retrieval is not
yet established. Although the removal of calcium ions may
cause protein structural modications to permit the physical
exposition of certain epitopes to antibody, it is likely that such
a mechanism is not universal.
Microwave procedures would cause a strong denaturation
of the secondary and tertiary structures of proteins (Dill and
Shortle, 1991). It is probable that the unmasking effect may be
due to thermal mechanisms as well as other mechanisms
induced by the molecular rapid oscillation (Stone et al., 1999).
Other authors stated that the mechanism of microwave antigen
retrieval is exclusively due to heat (Hopwood et al., 1988). On
the contrary, Porcelli et al. (1997) demonstrated that the
retrieving effect is caused solely by the microwave oscillating
electromagnetic eld. In any case, microwave irradiation can
alter the non covalent bonding of proteins, such as hydrogen
bonds, van der Waals and hydrophobic interactions (Leong and
Sormunen, 1998).
Since alcohol xation does not cause the formation of
crosslinks and, alcohol xed antigens can be also retrieved by
heat retrieval, other unknown mechanism may be involved in
the retrieving mechanisms by heat (Hayat, 2002).
It was speculated that antigen retrieval by ultrasound may
be due to the vibrations which make holes and gaps in the
biological structures (Podkletnova and Alho, 1993). As hypothesized by Chu et al. (2006), ultrasound would accelerate the
protein crosslinks formation during aldehyde xation. During
this formation, the involvement of the chemical intermediates
intervening in ordinary xation and highly denaturant, are
avoided. The fast crosslink formation would involve reactive
chemical groups different from those induced by a normal
xation, which cause the loss of antigenicity.
Regarding antigen retrieval by enzymatic treatment, the
mechanism would involve the disruption of the crosslinks
between the epitopes and the xative molecules, which have
modied the primary and tertiary structures of the protein
(Leenen et al.,1985), but not the secondary structure (Mason and
O'Leary, 1991). Since the success of such retrieving mechanism
depends on the number of lysine, arginine or asparagine
residues (Leenen et al., 1985), the enzymatic treatment can be
useful in retrieving proteins which contain such amino acids
residues (D'Alessandro et al., 2004). Some antigens may be
destroyed by this treatment (van Hecke, 2002).
Guhl et al. (1998) stated the mechanism of the enzymatic
digestion by N-glycanase F is probably based on the improved
accessibility to the glucidic epitopes.
The antigen unmasking can be also accomplished by
chaotropic agents, such as urea and guanidinium chloride.
These substances can disrupt molecular structure in which
hydrogen bonds, hydrophobic and Van der Waals interactions
are involved.
Recently, heat retrieval by citraconic acid has been introduced (Namimatsu et al., 2005). Briey, these authors have
postulated that citraconic acid would charge proteins negatively. The cooperation between the electrostatic repulsion and
the hydrophobic attraction would expose the antigens.
Detergents and denaturants do not seem to be able to reverse
the effect of aldehyde induced modications. Indeed, such
agents represent cofactors in retrieval process, because they are
able to render the proteins susceptible to the unmasking effect

by other primary factors (Fowler et al., 2007). The sodium
dodecyl sulphate unmasking effect is explained by the disruption of oligomerized structures of certain proteins, such as
caveolins, to permit the access of the primary antibodies to the
epitopes (Robinson and Vandr, 2001).
Alternative explanations for antigen retrieval mechanisms
exist. For example, extraction of diffusible proteins, precipitation, stabilization and rehydratation of protein epitopes may
be involved in unmasking (Suurmeijer and Boon, 1993a; Boon
and Kok, 1994; Shi et al., 1997). Moreover, antigen determinants located on the inner portion of antigen molecules or
hidden by other oligomeric components may also be exposed
by heat denaturation, even though tissues are not treated
with chemical xatives (Yasuda et al., 1986; Yamashita et al.,
1989, 1997; Robinson and Vandr, 2001).
Recently, the mechanism of antigen retrieval has been
explained by the involvement of Mannich reaction (Sompuram
et al., 2004, 2006a; Leong and Leong, 2007), where a reaction of
amino alkylation occurs. Sompuram et al. (2004) developed an
in vitro molecular model to study the sensibility of linear
epitopes to aldehyde xation. They found that antigen retrieval
efciency was dependent on the presence of a tyrosine residue,
located in the epitope or in neighbouring site, and on an
arginine located elsewhere. Such residues can form covalent
bond between them by Mannich reaction.
Sompuram et al. (2006b), using a peptide array model, have
found that protein antigen conformation was not important
for the epitope retrieval. Indeed, adjacent protein domains to
epitope represented the factor inuencing the retrieval. Sompuram et al. (2006a) stated that antigen retrieval by heat was not
able to reverse the protein conformation, because of its high
denaturant power. In addition, since protein primary structure is
not affected by xation, they hypothesized that linear epitopes
are responsible for the antibodyantigen interactions. The
mechanism of masking by xation could be explained by the
aldehyde-induced formation of a steric interference of the linear
epitope. Thus, antigen retrieval mechanism would lie in
removing such steric interference by adjacent protein domains.
Boenisch (2006) speculated that, in most cases, formaldehyde xation can also induce a variation of the electrostatic
charges of antigen, and that antigen retrieval would restore the
native electrostatic charges necessary for the initial antigen
antibody interactions. For the reason above, pH and ion
composition of antibody diluent are very important factors
affecting the staining efciency (Larsson,1988; Boenisch, 2001).
Finally, it is important to say that also other factors, as
alcohol dehydration may induce a loss of immunoreactivity,
whereas the use of xylene and hot liquid parafn do not seem
to alter the epitope conformations (Stein et al., 1985).
The mechanism of antigen retrieval for xed and parafn
embedded tissue can be also extended to the specimens
processed for electron microscopy. But, for epoxy embedded
tissue, additional explanations were given by Brorson (1998).
Since epoxy resins copolymerizes with the tissue (Causton,
1984), this author suggests that antigen retrieval disrupts
also the links between the tissue and the resin medium.
In acrylic sections there is no copolymerization between the
tissue molecules and resin medium (Kellenberger et al.,
1987). For this reason, acrylic sections are more suitable for
immunohistochemistry.
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ARTICLE IN PRESS
Table 3
A summary of the most common theories on mechanisms of antigen retrieval
Principle
Retrieval agent Author(s)
Hydrolytic breaking
of the intra- and
intermolecular crosslinks
Heat
Enzymatic
treatment
Renaturation of epitope
conformation
Removing of steric
interference by adjacent
protein domains
Heat
Heat
Disruption of links between Heat

tissue components and
the resin medium
Extraction, precipitation,
Heat
stabilization and
rehydratation of epitopes
Electrostatic restoration
Heat
of epitopes
Heat and
citraconic acid
Holes and gaps formation
Ultrasound
in the biological structures
Shi et al., 1991; Suurmeijer

and Boon, 1993a; Montero
et al., 2000; Rait et al., 2004;
Yamashita and Okada, 2005a;
Emoto et al., 2005
(Leenen et al., 1985; Mason
and O'Leary, 1991;
D'Alessandro et al., 2004)
Volkin and Klibanov, 1989
Sompuram et al. 2006b;
Yasuda et al., 1986; Yamashita
et al., 1989, 1997; Robinson
and Vandr, 2001
Brorson, 1998
Suurmeijer and Boon, 1993a;

Boon and Kok, 1994; Shi
et al., 1997
Boenisch, 2006
Namimatsu et al., 2005
Podkletnova and Alho, 1993
In Table 3 the most common theories on mechanisms of

antigen retrieval are summarized.
3. Retrieval efcacy
3.1. Overview
In general, antigen retrieval techniques, especially those
mediated by heat, are useful to unmask most of antigens.
Jasani and Rhodes (2001), studying the data of assessments
of the UK National External Quality Assurance Scheme
(NEQAS), observed that, for some abundant cytoplasmic
epitopes, a proteolytic digestion may be preferred, although
a heat-mediated retrieval may greatly enhance the detection
sensitivity.
Although the use of antigen retrieval is essential for some
epitopes, the staining for other ones can be enhanced.
Since antigen retrieval mechanism would mainly consist
in the cleavage of the xation-induced crosslinks and in the
subsequent structural reconstruction of epitope, all the
factors, which can contribute to this epitope reformation,
can play an important role in retrieval efcacy. In fact, it is
known that the success of antigen retrieval is determined by
several factors, which include pH, molarity, elemental nature
and concentration of the retrieval buffer used, clone and
dilution of primary antibody (Taylor et al., 1996a; Evers et al.,
1998; Miller et al., 2000; Boenisch, 2001; Kan et al., 2005).
Moreover, for heating approaches, temperature and irradiation time are also very important factors (Evers and Uylings,
1994; Shi et al., 1995a, 1996a, 2002).
Regarding the duration of storage time of parafn embedded
specimen blocks, it seems that the length of storage does not
cause a signicant problem with loss of antigenicity (Cattoretti

et al., 1992; Hann et al., 2001).
Different retrieval solutions can be used. Distilled water,
EDTA, aluminium chloride, zinc sulphate, lead thiocyanate,
formic acid, urea, citrate buffer, citraconic anhydride represent or have represented different solutions used according to
the antigens under study (Suurmeijer, 1992; Cattoretti et al.,
1993; Lucassen et al., 1993; Norton et al., 1994; Malisius et al.,
1997; Pileri et al., 1997; Namimatsu et al., 2005).
3.2. Fixation effect
Generally, prolonged as well as insufcient xation times
may affect the sensitivity of the immunohistochemical staining
(James and Hauer-Jensen, 1999; Werner et al., 2000; Hayat,
2002). Although, before the introduction of the antigen retrieval
technology, it was highly recommended to x the tissue for
the minimum requested time (Fox et al., 1985; Battifora and
Kopinski, 1986; Leong and Gilham, 1989), nowadays we know
that heat-mediated antigen retrieval can reduce or eliminate
the staining variations caused by prolonged formalin xation
(Shi et al., 1997). The staining patterns of some antigens, such as
the proliferating cell nuclear antigen, are relatively independent
of xation time (6 h to one week) (Haerslev and Jacobsen,1994).
Munakata and Hendricks (1993) showed that the optimal
xation time was 4 h for the labelling of Ki-67 by antigen
microwave retrieval. They concluded that for unknown xation
times, longer heating times were preferred to shorter times,
since long microwave treatments did not show negative effects
under their experimental conditions. However, extended
retrieval time can be potentially harmful to the tissue and
cause damage overretrieval. Lee et al. (2002) found that
different durations of xation (at least for 24 h) did not show
any adverse effect on oestrogen receptor immunohistochemistry performed with the same antigen retrieval protocol.
A study of Arber (2002) on effects of prolonged formalin
xation on the immunohistochemical reactivity of breast tissue,
showed that formalin xation did not signicantly reduce
immunoreactivity for Ki67, p27, or vimentin, even in tissue xed
for 154 days. Miller et al. (2005) showed that a xation period of
36 days did not affect adversely the immunocytochemical
staining of bovine viral diarrhea virus (BVDV). However, a
prolonged xation time (i.e. 137 days) caused false-negative
results. Hoetelmans et al. (2001) showed that after a prolonged
xation time the immunohistochemical nuclear staining of Bcl2 was irreversibly lost.
In any case, a complete and rapid xation minimizes false
localizations and antigen diffusion artifacts.
Unlike formaldehyde, glutaraldehyde has two functional
groups which cause the formation of very stable and irreversible crosslinks (Eltoum et al., 2001).
In a comparative study of archival tissue Hann et al. (2001)
found that specimens xed simultaneously in 4% paraformaldehyde and 2% glutaraldehyde showed a higher labelling
density in comparison to the specimens xed with lower
aldehyde concentrations. These authors hypothized that higher
concentration of xative can keep intact more antigenic
epitopes. Furthermore, they suggested that such a phenomenon
could be explained by the simultaneous presence of the two
aldehydes, as well as, by the different chemical xation
properties. In fact, it is known that the rst crosslinking action
Methods (2008), doi:10.1016/j.jim.2008.11.007
ARTICLE IN PRESS
6
is reversible and caused by formaldehyde. Subsequently, the

late action of glutaraldehyde would cause a displacement or
additional crosslinks of epitopes (Hayat, 1989).
3.3. Heat effect
The most important factor in retrieval efciency is represented by heat (Shi et al., 1991; Stirling and Graff, 1995). It was
demonstrated that optimal and efcient results are correlated
with the product of temperature and time of irradiation (Koopal
et al., 1998; Shi et al., 1998b). Most protocols recommend the
use of 1020 min of irradiation at about 100 C. However, many
authors recommend higher temperatures and irradiation times
longer than 20 min (von Wasielewski et al., 1994; Shi et al.,
1995a; Evers and Uylings, 1997; Brorson, 2004). For maximizing
the labelling intensity, the use of 10 mM citrate buffer, pH 6.0, at
135 C for epoxy sections (Brorson, 2002) or, the combination of
the irradiation at 144 C and of antibody incubation at 60 C for
epoxy gel antigen systems, were applied (Brorson, 2004).
Since high temperatures can cause strong background
staining (Taylor et al., 1996a; Jiao et al., 1999), and are protein
denaturants, many authors recommend the use of lower
temperatures, such as 80 C (Ezaki, 1996; Jiao et al., 1999). Shi
et al. (2002) showed that for some antigens, as cyclooxygenase-2, it is mandatory to use a temperature of 90 C instead of
the usual 100 C.
It is clear that heat duration and temperature is antigen
and antibody dependent.
3.4. pH effect
Although there is no universally optimal pH of the
retrieval medium, it has been shown that this factor is the
major critical one for a proper protein refolding (Evers and
Uylings, 1994; Shi et al., 1995b; Emoto et al., 2005). Although
some antigens can be retrieved at any pH, others prefer low or
high pH (Shi et al., 1995b).
It is clear that the pH plays an important role during the
epitope reformation, because of generation of electrostatic
repulsion and hydrophobic attractive forces, which would
contribute to the antigen/antibody reaction. Moreover, antigen retrieval process may be improved if acidic or basic
solutions are used. In fact, hydrolysis of amides is favoured in
acidic or basic solutions. Since the pH strength is higher for an
acid solution in comparison to a basic one, a basic solution
seems to be more efcient in retrieving antigens (for a more
detailed description, see Kim et al., 2004a).
It seems that there is no direct relationship between the
isoelectric point of the protein and the pH of the retrieval
solution used (Yamashita and Okada, 2005a).
The optimal pH is solution-dependent (Saito et al., 2003).
The use of citrate buffer at acidic pH, and TrisHCl at basic pH,
seem to be the most often used methods of retrieval for the
majority of antigens.
3.5. Metal ions effect
Initially, Shi et al. (1991) suggested that metal salts could
play a role in antigen retrieval efcacy. Subsequently, it has
been recognized that their role is not critical in the retrieval
mechanisms.
Regarding calcium ions, their presence may inuence the

immunostaining results for a limited number of epitopes.
A discussion on the effects and theories of calcium ions is
given in Section 2.
The retrieval efcacy can be modied by the addition of
NaCl to buffers. The role of NaCl is pH-dependent. In fact,
Emoto et al. (2005) hypothesized that, at pH 9.0 and in
presence of NaCl, the epitope electrostatic repulsion is
decreased, and thus the hydrophobic forces cause the epitope
entangling. On the contrary, at pH 10.5, the salt-dependent
reduced electrostatic repulsion could moderate the denaturation of negatively charged epitope portions.
3.6. Solution type effect
Stirling and Graff (1995) suggested that the application of
heat, rather than the composition of the retrieval medium, is
the principal factor in unmasking process. However, such a
chemical composition may represent a cofactor in heatmediated approaches. Kim et al. (2004a) showed the importance of such a factor. In fact, at constant pH value, different
buffers produced different immunolabelling intensities. The
authors hypothesized that these results may be related to the
minor interactions of buffers with proteins and metals. On
the contrary, Katoh and Breier (1994) showed that immunostaining of p53 could be obtained using indifferently citrate
buffer, distilled water or normal saline.
In Section 2 we have discussed in detail the role of buffer
solutions containing EDTA or EGTA in chelating calcium. Such
solutions can increase efcacy of antigen retrieval only for a
very limited number of antigens.
3.7. Molarity effect
Initially, the molarity of retrieval solution was recognized
as an important factor for the retrieval efcacy (Cattoretti
et al., 1993). Subsequently, this variable was found to be less
important (Shi et al., 1997; Kanai et al., 1998). However, the
importance of molarity is solution-dependent. Suurmeijer and
Boon (1993b) found that the optimal immunolabelling for
vimentin was obtained with the use of 4% aluminium chloride.
3.8. Non specic background
It has been shown that antigen retrieval techniques have
potential limitations and drawbacks (Shin et al., 1994; Baas
et al., 1996; Ezaki, 1996; Kodama et al., 1997; Allison and Best,
1998). One of the most important problems in antigen retrieval
is the non-specic background stainings (Ho et al., 1994; Kim
et al., 2002).
Although the phenomenon is not universal, there is a
general correlation between the increased non-specic background and the raising of temperature in heat-mediated
antigen retrieval (Ezaki, 1996). Although the severity of the
antigen retrieval conditions is proportional to length and nature
of xation (Battifora and Kopinski, 1986; McNicol and Richmond, 1998), an overtreatment of the antigen retrieval may
produce an excessive change in protein conformation. Therefore, the heating effect in antigen retrieval is proportional to the
product of time and temperature (Kawai et al., 1994; Shi et al.,
1995a).
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ARTICLE IN PRESS
An increased background can be also obtained with the use

of solutions of very high or low pH values (Kim et al., 2004a).
Such undesired background stainings may be induced by nonspecic hydrophobic and ionic interactions between antibodies
and denatured epitopes. However, we can not exclude that
such an effect is caused by the use of a non-optimal antibody
concentration. In fact, with the increasing of the sensitivity of
the method introduced by the antigen retrieval, it is necessary
to adjust again the optimal antibody concentrations (Shi et al.,
1997).
An overtreatment with enzymatic digestion can cause a
decreased staining as well as a false negative, because of the
protein denaturation by such enzyme digestion (Cattoretti
et al., 1993).
It is known that background staining can be produced by
hydrophobic interactions of proteins as well as ionic and
electrostatic interactions. Since antigen retrieval itself can alter
these processes, if inappropriately applied, such approaches can
cause false positive, negative or nonspecic background stainings. A further explanation for the occurrence of false positive
stainings, is that antigen retrieval may also expose cross-reactive
epitopes (Nyhlin et al., 1997). In conclusion, it is important to
understand the mechanisms, as well as the limitations of antigen
retrieval methodology, if we want to avoid of introducing false
positive or negative results into our experiments (Ezaki, 2000).
Retrieval efciency may depend also on detection system
of antibodies. The avidinbiotin complex (ABC), after antigen
retrieval, can cause the non-specic staining. It is known that
detection system can unspecically bind with endogenous
lectins, sugars and specically with endogenous biotin (Duhamel
and Whitehead, 1990; Houen and Hansen, 1997; Nyhlin et al.,
1997; Kim et al., 2002). The presence of endogenous biotin
may represent a relevant source of non-specic staining when
ABC methods are used in combination with high temperature
retrieval techniques. In fact, antigen retrieval can unmask the
endogenous biotin (Dodson, 2002; Mount and Cooper, 2001; Kim
et al., 2002). In order to avoid such false positive results, a battery
of appropriate controls must be performed as well as blocking of
endogenous biotin by a sequential incubation of tissue sections
with avidin and subsequently with biotin, or alternatively a
different non avidin biotin based detection system can be used
(Wood and Warnke, 1981; Kim et al., 2002; Dodson, 2002).
4. Light microscopy application
4.1. Enzymatic digestion
Introduced in the 1970s and still used for certain antigens,
proteolytic induced epitope retrieval (PIER) consists of
the controlled treatment of tissue section with proteolytic
enzymes (Huang, 1975; Huang et al., 1976; Curran and
Gregory, 1977; Mepham et al., 1979; Battifora and Kopinski,
1986; Ordonez et al., 1988).
Enzymatic digestion is used to retrieve epitopes which
may loose their antigenicity with heat-based methods. This
approach is now used for a limited number of antigens, such
as cytokeratins (Jasani and Rhodes, 2001). Unfortunately, the
use of enzymatic digestion is a more aggressive approach in
comparison to the methods using heat. In fact, it can destroy
some epitopes as well as the tissue morphology (Pileri et al.,
1997), if used for extensive time.
In general, dewaxed and rehydrated sections are treated with

a proteolytic enzyme at 37 C, or at room temperature. In order
to limit the enzymatic reaction to the xation-induced bonds,
experimental factors, as concentration, time, and temperature
must be controlled. Subsequently, the enzymatic reaction is
blocked with the use of a cold buffer at +4 C (Shi et al., 1993).
The use of proteinase K represents an efcient approach
for the retrieval of some antigens (Rojo et al., 2006; Jessie
et al., 2004). Other proteases, such as trypsin (0.05% w/m in
PBS plus 0.1% CaCl2), chymotrypsin, pepsin (0.05% w/m in 2N
HCl), pronase (0.05% w/m in PBS) are used for uncovering
antigen sites (Ward et al., 2006).
Some recent examples of application of enzymatic digestion are discussed below.
Collagens can be retrieved by proteolytic digestion.
Wakamatsu et al. (1997) suggested the use of pepsin (1 mg/ml)
in hydrochloric acid, pH 2.0, for 3060 min at 37 C, for
immunostaining of type III and IV collagens. A similar approach
was suggested by Franciosi et al. (2007) for the optimal
localization of brain vascular collagen VI.
Takada et al. (2000) showed that trypsin treatment (0.1%,
at 37 C for 30 min) was the best antigen retrieval approach to
immunolocalize opsin antigens in parafn tissue sections of
rabbit retina. Treatment with proteinase K or microwaving
failed to produce optimal staining because they impaired
tissue morphology or resulted in high background of staining,
respectively.
Guhl et al. (1998) introduced a new method for antigen
retrieval in light and electron microscopy. They pre-treated,
prior to immunolabelling, parafn and ultrathin cryosections
with N-glycanase F (0.25 U/ml in PBS, pH 7.4, overnight at 37 C)
in order to remove N-glycosidically linked oligosaccharides.
The authors improved signicantly the immunoreaction for
poly 2,8 KDN (KDN, 2-keto-3-deoxy-D-glycero-D-galactonononic acid) of megalin, a highly glycosylated protein. The
mechanism of retrieval would involve the removal of sterical
hindrance by large N-glycosidically linked oligosaccharides. For
ultrastructural immunolocalization, the same authors obtained
a more efcient labelling of some membrane proteins with the
exposure of cryosection to 0.5 U/ml N-glycanase F in phosphate
buffer, pH 5.5, for 4 h at 37 C. These authors suggested that the
mechanism of antigen retrieval for cryosections exposed to
acidic pH was dependent on the depolymerisation of methylene
and polymethylene bridges introduced by formaldehyde in the
acidic environment.
For extracellular epitopes, such as collagens, bronectin
and proteoglycans, hyaluronidase treatment was successfully
applied (Holund and Clemmensen, 1982; Lukinmaa and
Waltimo, 1992; Sabit et al., 2001).
Nucleases, such as deoxyribonuclease has been successfully used to immunostain cancer tissues for the estrogen
receptor with H-222 monoclonal antibody (Shintaku and
Said, 1987). However, if other types of antibodies, such as the
monoclonal ID6 and D12, are applied, the heat approaches
yield best results (Yamashita, 2007).
4.2. Heat
Nowadays, heat induced antigen retrieval is the most
commonly used technique (Boon and Kok, 1994; Shi et al.,
1997; Werner et al., 1996; Leong and Sormunen, 1998; Jasani
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ARTICLE IN PRESS
8
and Rhodes, 2001, for reviews). Introduced in the early 1990s

(Shi et al., 1991, 1992), such an approach can be successfully
applied to a wide range of antigens xed with formaldehyde
xatives.
Cuevas et al. (1994), with the use of microwave irradiation,
retrieved 20 different antigens, that before could only be
demonstrated on frozen sections.
In order to heat tissue sections, several methods can be
used: microwave (Shi et al., 1991; Cattoretti et al., 1993),
autoclave (Shin et al., 1991; Bankfalvi et al., 1994), pressure
cooker (Igarashi et al., 1994; Eagle et al., 1997; Norton et al.,
1994), steamer (Suurmeijer and Boon, 1993b) and water bath
(Shi et al., 1991; Kawai et al., 1994).
In general, although the chosen heat source is indifferent
(Taylor et al., 1996a), the microwave oven represents the most
common heat source. However, microwave treatment shows
some drawbacks, such as the generation of artifacts caused by
the evaporation of the buffer or local superheating (Norton
et al., 1994). Moreover, contrarily to autoclave, microwave use
may impair cell morphology (Hunt et al., 1996). Norton et al.
(1994) suggested that the microwave oven can be replaced by
a domestic pressure cooker, improving the efciency of the
method in terms of time and costs.
A commonly used method involves the microwave
heating of dehydrated parafn-embedded tissue in 10 mM
citrate buffer with a microwave oven at 100 C for 510 min
(Chiu, 1987; Shi et al., 1991; Cattoretti et al., 1992; Brown and
Chirala, 1995).
Slight modications of this approach are used. For
example, Nyhlin et al. (1997) succeed in retrieval several
human duodenal endocrine antigens by microwave heating in
10 mM citrate buffer, pH 6, for three boiling cycles of 5 min
each.
In order to enhance the immunoreactivity of prion
proteins, Furuoka et al. (2005) showed that raising temperature to +135 C in autoclave was the best approach, but only
for antibodies recognizing the linear epitope.
Although citrate buffer represents the most commonly
used medium, other saline solutions were used, such as lead
thiocyanate, zinc sulphate, and aluminium chloride (Boon and
Kok, 1994).
Evers and Uylings (1997) showed that the best approach
for the retrieval of several neuronal antigens was the use of
microwave pretreatment of tissue blocks in Tris Buffered
Saline (TBS), pH 9.09.5.
As stated above, calcium ions may play an important role in
epitope masking (Morgan et al., 1994, 1997). For this reason,
the addition of chelating agents, such as EDTA and EGTA, to
retrieval solutions, improved the retrieval efcacy for certain
antigens. Pillai et al. (2003) showed that the nuclear localization of the tumour suppressor p53 in normal tissues with the
D07 antibody, was possible only following an EDTA-based
retrieval approach consisting of freshly prepared buffer
consisting of 0.1 M EDTA with 0.1% Tween pH 8.0. Kim et al.
(2004b) found that borate (pH 8.0) and Tris (pH 9.5)
represented the best retrieval solution choice for the immunohistochemical visualization of 29 common antibodies.
The use of citraconic anhydride solution (0.05% citraconic
anhydride solution, pH 7.4, at 98 C for 45 min) has been
introduced by Namimatsu et al. (2005), who retrieved
efciently many human antigens. These authors showed that
parafn sections, treated with this method, exhibited similar

immunolabelling patterns to those of fresh frozen sections.
Recently, Long and Buggs (2008) have reported a rapid
microwave-based technique for immunouorescent staining
of several antigens in formalin xed and parafn embedded
tissue. The protocol reduces the incubation times of the
antibodies, and avoids the use of blocking reagents.
4.3. Heat and enzymatic digestion combination
Alternatively, if a simpler method fails to retrieve antigen
sites, a combination of heat and enzymatic digestion can be
used. This approach is useful when a multiple labelling is to be
performed.
Merz et al. (1993) suggested a combination of protease
digestion and microwave treatment in order to improve the
staining of surface and cytoplasmic immunoglobulin heavy
and light chains. However, Cuevas et al. (1994), in a combined
approach with microwave irradiation and trypsin digestion
for the retrieval of several antigens, did not show a better
staining quality achieved with microwave treatment alone.
Ezaki (2000) suggested a combination of mild microwave
heating (in 10 mM citrate buffer, pH 6.0 at 80 C, for 1520 min)
and pepsin digestion (0.005% in 10 mN HCl, pH 2.0, at 37 C for
15 min), for proliferating cell nuclear antigen (PCNA) visualization on formaldehyde-xed and parafn embedded rat tissue
sections, as well as, for double immunostaining of PCNA
together with -actin, bromodeoxyuridine, keratin, type IV
collagen and vimentin. A similar suggestion was given by Frost
et al. (2000) to immunostain estrogen and progesterone
receptors in breast carcinomas. Sections were retrieved in
10 mM citrate buffer at 80 C for 2 h following trypsin treatment.
4.4. Ultrasound
Ultrasound is mainly used to minimize the xation-induced
protein alterations. Thus, this approach is aimed at preserving
the molecular antigenicity, rather than in unmasking it.
Podkletnova and Alho (1993) increased considerably the
sensitivity of immunouorescence staining with the simultaneous use of 1020 s ultrasonic irradiation and incubation
with primary antibody for the visualization of a neuropeptide
diazepam binding inhibitor and glial brillary acidic protein
in free-oating sections.
Chu et al. (2005) used a formalin xation procedure
combined with high-frequency and high-intensity ultrasound
for 15 min. They showed that such approach not only reduced
xation time, but also resulted in better preservation of
morphology and immunoreactivity.
However, Hammoud and Van Noorden (2000) have found
that the use of ultrasound treatment did not improve the
antigen retrieval level reached with the microwave-based
approaches. Similar conclusion was obtained by Leong et al.
(2002), who stated that ultrasound treatment did not give
consistent and better results in comparison to alternative
antigen retrieval methods.
4.5. Minor approaches
Many other approaches exist to retrieve different antigens.
Some examples of such treatments are discussed below.
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ARTICLE IN PRESS
Antigen retrieval was carried out simply by immersing

tissue in water, or 10% sucrose in PBS for different incubation
times (Puchtler and Meloan, 1985; Elias, 1990). The unmasking effect would lie in the slow hydrolysis which would break
the aldehyde induced crosslinks (Helander, 1994).
Protein denaturants, such as urea, guanidine hydrochloride,
guanidine thiocyanate, formic acid have also been successfully
used (Hausen and Dreyer, 1982; Shi et al., 1993, 1996a;
Beckstead, 1994; Ho et al., 1994; Privat et al., 2000). Formic
acid has been used in neurolament (Cammarata et al., 1990)
and prion proteins retrieval (Kitamoto et al., 1987; Doi-Yi et al.,
1991). The combination of formic acid and heat has been used in
retrieving antigen involved in amyloid deposits (Haritani et al.,
1994; Cummings et al., 2002). Malisius et al. (1997) found that
the combination of microwave and 0.1 M formic acid in 2%
gelatine for 1 min is the best treatment for the immunohistochemical demonstration of CD2, CD3, CD4, and CD5.
The chemical detergent Triton X-100 has been used to
retrieve some antigens. Gutierrez et al. (1999) incubated
sections in 0.25% Triton X-100 in TBS for 30 min at room
temperature.
Peston and Shousha (1998) utilized boric acid, as well
as acetic acid (0.2 M. pH 7.0), to immunolocalize estrogen
and progesterone receptors. Similarly, Wilson et al. (2007)
successfully retrieved CD31 and proliferating cell nuclear
antigens with the use of boric acid.
Antigen retrieval by hydrogen peroxide treatment (510% in
PBS, for 30 min) improves the labelling efciency in immunoperoxidase staining for some drugs, such as daunomycin,
bleomycin and pepleomycin (Ohara et al., 2007). In vivo these
drugs undergo a chemical reduction transforming themselves
into semiquinone and/or hydroquinone derivative. The oxidation by H2O2 seems to transform such derivatives into the
original form.
Recently, Wong et al. (2007) retrieved successfully several
tumour antigens with the use of bifunctional SkipDewax,
which simultaneously dewaxes parafn slides. The treatment
was performed in a pressure cooker for 2.5 min.
Shi et al. (1992) used a methanolic solution of NaOH for
retrieving antigens in formalin-xed and celloidin-embedded
bone sections. For celloidin-embedded bone sections Shi
and Tian (1993) used a combined approach of heating and
NaOH-methanol treatment. A similar combined approach was
used by Shi et al. (1998a). A combined exposure of celloidin
embedded sections to NaOH solution and trypsin digestion
was successfully used by Ganbo et al. (1997). Hydroxides were
also used by Mukai et al. (1992), who used KOH in 70% ethanol
to retrieve actin domains in parafn sections.
4.6. Approaches for non embedded specimens and free oating
sections
Antigen retrieval techniques can also be applied for the
immunohistochemistry of many antigens in non embedded
specimens. Many procedures have been performed before or
after sectioning.
To visualize -glutamyl transpeptidase, xed frozen sections were simply heated in PBS at 95 C for 10 min (Yamashita
et al., 1989).
Before obtaining vibratome free oating unfrozen sections,
xed brain slices were successfully treated with microwave in
TrisHCl, pH 9.0, at 95 C for 15 min (Evers and Uylings, 1994,

1997).
Shiurba et al. (1996, 1998) microwave treated free oating
vibratome sections in 50 mM citrate buffer, pH 6.0, at 95 C for
5 min in order to unmask brain tau phosphoserine 413 and
tau protein kinase I.
Ino (2003) developed a simple method for a wide range of
antigens of mouse brain and rat testis aldehyde-xed frozen
sections. Antigen retrieval was performed before sectioning
and freezing. After paraformaldehyde xation, tissue specimens were immersed in distilled water or 10 mM sodium
citrate buffer, pH 6.0, at 4 C overnight. After the incubation
time, tissue blocks were heated at 90 C in the previous
retrieval solution for 3 min. Subsequently, pieces were placed
in cold PBS containing 30% sucrose at 4 C for 24 h. Finally,
tissue blocks were frozen and sectioned.
Yamashita and Okada (2005b) used a new antigen retrieval
approach to aldehyde xed fresh frozen sections. After xation
with formalin containing CaCl2, the frozen sections were
autoclaved in 20 mM TrisHCl buffer (pH 9.0) for 10 min at
120 C. This approach is useful when soluble antigens are
extracted from fresh frozen sections during xation. They also
showed that the use of EDTA in retrieving approaches was
inert on immunolabelling efciency.
It is not easy to apply heat-mediated antigen retrieval to freeoating sections, because of damage to sections. To overcome
such an obstacle, Jiao et al. (1999) described a simple method for
free oating and xed sections or slide mounted xed cryostat
sections. They recommended a water-bath heating at 80 C for
30 min in 1050 mM sodium citrate (pH 8.59.0).
Among protein denaturants, sodium dodecyl sulphate,
guanidine hydrochloride and urea have been used for antigen
unmasking in frozen or unfrozen specimens. Frozen sections
were treated successfully with 6M urea or in 6M guanidine
hydrochloride in 20 mM PBS, pH 7.4, at room temperature, for
15 min (Yamashita et al., 1989). In order to visualize several
antigens of Semliki Forest virus, Peranen et al. (1993) exposed
xed and permeabilized cell culture to 6N guanidine hydrochloride (in 50 mM TrisHCI, pH 7.5, for 10 min). Sodium
dodecyl sulfate (SDS) treatment was used by Soltys and Gupta
(1992) in xed cell cultures to immunolabel the heat shock
protein HSP 60. Similarly, glutaraldehyde-xed cultured cells,
as well as frozen section, were treated with SDS to improve
the staining efciency for tubulins and caveolins (Robinson
and Vandr, 2001). Brown et al. (1996) retrieved some
antigens (Na/K-ATPase, AE1/2 anion exchanger, caveolin,
rab4 in cryostat sections with the use of 1% SDS for 5 min. A
similar approach was carried out by other authors for the
immunohistochemical demonstration of certain microtubule
proteins (Ding et al., 1995; Robinson and Vandr, 1997), and
other protein components (Brown et al., 1996). Fowler et al.
(2007) in an articial protein gel-system, for preteomic study
purposes, found that the best retrieval for lysozyme was the
combination of heat, glycine and 2% SDS.
5. Electron microscopy application
5.1. Some considerations for electron microscopy
In transmission electron microscopy, osmium tetroxide, used
as secondary xative, and plastic resins, used as embedding
Methods (2008), doi:10.1016/j.jim.2008.11.007
ARTICLE IN PRESS
10
media, represent additional impairing factors for immunocytochemical reactions.

The antigen retrieval in osmium tetroxide xed specimens
may be performed with the use of bleaching techniques,
which make use of oxidizing agents (Bendayan and Zollinger,
1983). Sometimes this approach does not sufce, suggesting
that the reaction of osmium tetroxide with antigen is not
reversible, and such a reaction may inuence also the tissue
penetrability (Bendayan et al., 1987; Roth, 1982).
Hydrophobic plastic resins, such as the epoxy one, cover the
antigenic sites, because of its interactions with specimen
components, such as amino-, carboxyl- and sulphydryl groups
(Stacey et al., 1958). Moreover, the tissue is embedded in the
resin hydrophobic polymeric network, which prevents the
penetration of antibodies to the antigenic sites (Horobin, 1983).
For this reason, large proteins are very difcult to immunolabel,
in comparison to smaller antigens (Ottersen, 1989).
In order to minimize the above problems for immunocytochemical purposes, polar hydrophilic embedding media
have been introduced (Kellenberger et al., 1987; Newman,
1989; Merighi and Polak, 1993). In any case, for some antigens,
retrieval techniques are still required (Xiao et al., 1996).
Alternatively, but less efciently than the use of polar
hydrophilic embedding media, several techniques, collectively
named etching techniques, may be used to remove the
embedding media. The deplasticization of these methods
allows more epitopes to be exposed to the antibodies (Brorson
and Skjrten, 1995). However, such treatments may destroy
sensitive antigens (Baigent and Muller, 1990; Mar and
Wight, 1988).
5.2. Enzymatic digestion
Enzymatic digestion was also successfully used in resin
sections. These methods will be discussed in the Section 5.5,
because they consist of a combination of different approaches.
5.3. Heat
As for light microscopy, heating procedures can be used for
antigen retrieval and/or enhancing the immunolabelling
intensity for electron microscopy. The mechanism is the
heat-induced breaks of chemical bonds between the resin and
antigen molecules (Brorson, 1998, 2001a).
In order to retrieve antigen by heat, a microwave oven can
be used (Stirling and Graff, 1995; Rangell and Keller, 2000).
Heating can be obtained at temperatures below the boiling
point of water (Stirling and Graff, 1995) or, alternatively, at
higher temperatures (135 C) with autoclave (Brorson and
Nguyen, 2001).
Acrylic glycol methacrylate, epoxy and methyl methacrylate
resin sections have been treated successfully in 10 mM citrate
buffer, pH 6.0 (Suurmeijer and Boon, 1993a; McCluggage et al.,
1995; Hand et al., 1996; Rangell and Keller, 2000; Brorson,
2001b; Brorson and Nguyen, 2001; Groos et al., 2001).
Heating by microwave may cause some problems, such as
destruction of thin sections caused by bubble formation (Saito
et al., 2003). In order to overcome such problems, Saito et al.
(2003) submerged their grids in the retrieval solution, rather
then oating them, and found that the best conditions for
retrieving the heat-stable O-antigen lipopolysaccharide (LPS)
were Tris at 65 C overnight. Alternatively, the use of a

computerized microwave, without boiling temperature, can
retrieve antigens for satisfactory electron microscopy visualization (Stone et al., 1999).
Yano et al. (2003) improved considerably the immunolabelling efciency for chromogranin A, in epoxy embedded
and osmium postxed sections, by microwaving in alkaline
solution (pH 10) with subsequent immunostaining at 60 C.
Alternative attempts, such as etching with sodium metaperiodate or microwave irradiation in citrate buffer, pH 6.0 or in
1 mM EDTA, pH 8.0, failed in retrieving antigens.
Fossmark et al. (2005) have found that the best combination for chromogranin A retrieval, in osmium tetroxide-xed
epoxy sections, was autoclave heating at 135 C in alkaline
solution.
Unfortunately, heat-treated acrylic sections can detach
from unsupported grids. However, in order to overcome such
an obstacle, some precautions can be taken for ultrathin
acrylic sections mounted on mesh grids (Xiao et al., 1996;
Comer et al., 1999). Goode et al. (2004), in order to avoid the
loss of acrylic sections, developed a retrieval approach
consisting of a slight modication of method of Shi et al.
(1995b) and Stirling and Graff (1995). Grids with LR Gold resin
sections were immersed for 10 min in 0.1 M sodium citrate
buffer, pH 6.0, at 95 C.
Similarly to the method of Stirling and Graff (1995), Hann
et al. (2001) boiled LR White sections in 10 mM citrate buffer
(pH 6.0, for 10 min on a hotplate) for the immunolocalization
of collagen IV and bronectin. Sormunen and Leong (1998)
successfully retrieved cytokeratin, vimentin, type IV collagen,
and -catenin by applying microwave (in citrate buffer,
pH 6.0) to LR White ultrathin sections.
Xiao et al. (1996) have successfully retrieved cytokeratin in
hepatocytes by heating Lowicryl K4M-embedded sections in
citrate buffer (pH 6.0) or EDTA solution (pH 8.0).
Although the gold pattern was distorted in comparison to
non retrieved sections, Solberg et al. (2006) improved the
labelling efciency for the nucleobindin and osteoadherin
proteins by heating (in 10 mM citrate buffer, pH 6.0, at 95 C
for 15 min) Lowicryl HM23 sections of paraformaldehydexed bone.
Rangell and Keller (2000) applied microwave antigen
retrieval in acrylic resin embedded sections as well as in
frozen section and found that the labelling density in both
section types was similar.
5.4. Chemical bleaching and etching
In order to restore antigenic sites after osmium tetroxide
xation, the application of oxidizing agents can be useful.
Treatments of sections with hydrogen peroxide (Moriarty,
1973; Roth, 1986; Holm et al., 1989) or with aqueous solutions
of sodium metaperiodate (Silva, 1967; Merriam, 1958;
Merighi, 1992; Stirling and Graff, 1995; Lobo et al., 2002)
may be helpful. Such treatments, other than restoration of the
epitopes blocked by osmium tetroxide, would enhance also
the hydrophilic nature of the embedding medium (Causton,
1984). Moreover, Pfeiffer (1982) stated that the oxidation by
hydrogen peroxide can disrupt epoxy resin bonds.
The immunolabeling of epoxy sections can be intensied
by using methods which dissolve the epoxy resins araldite
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ARTICLE IN PRESS
(Mayor et al., 1961; Berkowitz et al., 1968; Grube and

Kusumoto, 1986) and Epon (Lane and Europa, 1965; Erlandsen
et al., 1979; Iwadare et al., 1990; Vidal et al., 1995).
Such etching approaches involve the use of sodium
ethoxide or methoxide (Litwin et al., 1987; Schwendemann
et al., 1982; Wiley et al., 1987; Mar and Wight, 1988; Bettica
and Johnson, 1990; Baigent and Muller, 1990; Groos et al.,
2001).
Vidal et al. (1995) obtained successful immunostaining
of growth hormone and prolactin by treating epoxy sections
with Maxwell's solution of potassium hydroxide in absolute
methyl alcohol and propylene oxide (Maxwell, 1978) and
subsequently with 4% hydrogen peroxide or a saturated
aqueous solution of sodium metaperiodate.
5.5. Different approaches combinations
For epoxy embedded sections, the combination of etching
by sodium metaperiodate or ethoxide, and heating in citrate
buffer, was superior to either etching or heating alone (Stirling
and Graff, 1995; Groos et al., 2001). Brorson et al. (2001)
observed that although heating epoxy sections in citrate buffer
gives superior results to hydrogen peroxide treatment alone, a
combination of the two procedures is not better because the
labelling density is the same as in heat treated sections.
Bettica and Johnson (1990) labelled extensively glial
brillary acidic protein (GFAP) in osmicated and epoxy
sections, with a combined section pretreatment with
dilute sodium ethoxide and the subsequent use of sodium
metaperiodate.
Litwin et al. (1984) localized rat liver peroxisomal
enzymes in semithin epoxy sections by removing the resin
with sodium ethoxide and, by treatment of the sections with
proteases and, subsequently with oxidants.
Rcken and Roessner (1999) applied different combinations of etching/bleaching (3% H2O2, 10 min, at room
temperature; sodium metaperiodate, pH 6.0, 30 min, at
91 C) and antigen retrieval techniques (heating in H2O2 for
30 min, at 91 C; heating in 1 mM EDTA, pH 8.0, for 30 min,
at 91 C or in sodium citrate) for the immunotyping of
amyloid deposits in epoxy embedded specimens. Unfortunately, a good antigen retrieval approach/etching combination was often associated with an increased background
staining.
D'Alessandro et al. (2004) used successfully a combination
of etching (10% NaOH in absolute ethanol for 12 min),
bleaching (saturated aqueous solution of sodium metaperiodate for 10 min) and, proteolysis (0.10.4 mg/ml proteinase
K for 1 min) of sections for the immunolocalization of RhoA in
epoxy and osmicated semithin sections.
For the immunodetection of aquaporin-1, -2 and megalin,
Zhai et al. (2007) etched epoxy semithin sections with
methanolic potassium hydroxide, followed by microwave
heating.
Haraguchi and Yokota (2002) used a combination of
approaches for immunouorescence staining of different rat
kidney and liver enzymes, such as leucine aminopeptidase,
catalase, 3-ketoacyl-CoA thiolase, cathepsin D. This consisted
of removing the epoxy medium with 10% sodium ethoxide;
then the sections were treated with 0.05% trypsin and, nally,
with sodium borohydride.
11
5.6. Minor approaches

Sodium borohydride treatment (1% for 30 min) was used
for the immunolocalization of glutamate decarboxylase and
tyrosine hydroxylase (Kosaka et al., 1986).
Jiao et al. (1999), after water-bath heating of vibratome
slices in 10 mM sodium citrate (pH 8.5, at 7680 C for 30 min),
have successfully immunolocalized the vesicular monoamine
transporter-2 by treating sections with 1% sodium borohydride in PBS.
Such a treatment would cause: a) the restoration of
antigenicity by re-establishing the protein tertiary structure
with the transformation of glutaraldehyde-induced double
bonds in single ones; b) increased permeability to reagents,
and c) decreased background staining by reducing aldehyde
groups which have not reacted (Kosaka et al., 1986; Priestley
et al., 1992).
Incubation of sections with 2% SDS for 15 min was applied
to visualize glial brillary acidic protein (DeArmond et al.,
1981). Similarly, Rajamannan et al. (2002) succeed in retrieving caveolin 1 with the use of 1% SDS in osmium-xed and
Spurr-embedded sections.
In order to reduce the co-polymerization between antigen
proteins and epoxy medium, resin mixture modications are
shown to be useful in recovering some antigens (Brorson,
1996, 1998; Brorson et al., 1999a,b). The immunolabelling of
large proteins in epoxy sections can be intensied by using
more accelerator in the inltration and embedding of the
tissue, eventually in combination with heating the sections
(Stirling and Graff, 1995; Brorson and Skjrten, 1996; Brorson
et al., 1997; Brorson, 1998). Brorson (1998) showed that the
combination of increased amount of accelerator in the epoxy
medium and the retrieval by heating in citrate buffer is a
powerful approach for increasing immunolabelling on epoxy
sections. With this method, an immunolabelling for IgG
and brinogen in epoxy sections was more intense than in
sections embedded in the acrylic resin LR White.
6. A brief note on standardization
Immunohistochemistry is extensively used for diagnostic
and prognostic purposes, and its standardization is critical for
reproducible and reliable results. Immunohistochemistry can
be adversely affected by several factors, including preanalytic,
analytic and postanalytic, resulting in poor reproducibility,
variable consistency and interlaboratory variability (Goldstein
et al., 2007).
Despite enormous difculties, signicant efforts are being
made in attempt to standardize diagnostic immunohistochemistry and several suggestions, solutions and guidelines
regarding xation, processing, retrieval and analysis were
made (Battifora, 1988; Shi et al., 1998b; Werner et al., 2000;
O'Leary, 2001; Seidal et al., 2001; Leong and Leong, 2006;
Taylor, 2006; Goldstein et al., 2007).
Important components of standardization are also intraand interlaboratory programs, which assess daily control/
quality assurance measures (QC/QA) as well as external quality
assurance programs (EQA) ensuring quality results for patient's
care (Maxwell and McCluggage, 2000; Rhodes, 2003).
Although the introduction of more sensitive reagents
and detection systems in conjunction with automation, has
Methods (2008), doi:10.1016/j.jim.2008.11.007
ARTICLE IN PRESS
12
considerably increased the reproducibility and consistency of

immunohistochemical techniques, standardisation still lags
behind (Leong and Leong, 2006).
Antigen retrieval contributes signicantly to the performance of immunohistochemistry and is critically dependent
on the type of xative and length of xation. If properly
applied, antigen retrieval represents an useful contribution to
the standardization of immunohistochemistry (Boenisch,
2005).
Tissue specimens should be xed in 10% formalin in PBS,
neutral pH for at least 8 h (Goldstein et al., 2007). Unfortunately,
for many specimens the duration of xation is unknown,
resulting in under and overxed tissues. In general, underxation, as well as delay in xation, represents a more serious
problem than overxation (Werner et al., 2000; De Marzo et al.,
2002). Delay in xation may cause loss of immunoreactivity, as
well as production of an aspecic binding of antibodies to
unrelated antigens. It is very difcult to nd an optimal antigen
retrieval protocol for underxed tissues.
The adverse effects of a prolonged aldehyde xation can be
counterbalanced by a stronger retrieval approach (Battifora
and Kopinski, 1986; McNicol and Richmond, 1998). Thus, for
optimal results, experimental factors of antigen retrieval assays
must be adjusted according to the length of xation, as well as
to the characteristics of the antibody used (Munakata and
Hendricks, 1993; von Wasielewski et al., 1994; Miller et al.,
2000; Taylor, 2006).
Although a successful antigen retrieval protocol depends

on the ne-tuning of its physico-chemical parameters, such
an assay should remain in a small range that guarantees
optimal results. In fact, under- or over-retrieval can produce
several artifacts. Insufcient unmasking can result in poor or
false-negative results, whereas over-retrieval can produce
false-positive staining, non specic background, hole formation in sections, loss of tissue morphology and detachment of
the tissue sections (Ezaki, 1996).
For unknown conditions of xation, such as archival
specimens, or for the assessment of new primary antibodies,
the use of the test battery (Shi et al., 1996b, 1997) can be
very useful. In brief, the new primary antibody is applied to
slides which have been retrieved with different combinations of temperature and pH values.
The use of other types of xatives, such as ethanol and
acetone, is strongly discouraged. Many studies have shown
that several antigens can be lost, partially or completely,
following xation in acetone or ethanol (Yamashita and
Okada, 2005b). Although some antigens in ethanol-xed
specimens, as well as in unxed frozen tissue, can benet
from antigen retrieval (Itoh et al., 1995; Kakimoto et al., 2004),
most antigen retrieval approaches are not helpful for specimens xed with coagulant solutions. Formalin xation,
followed by an optimal antigen retrieval protocol, remains
the best choice for detection of many antigens (Shidham et al.,
2003; Shi et al., 2008).
Table 4
Some advantages and disadvantages for different antigen retrieval approaches are summarized
Retrieving method
Source
Advantage
Disadvantages
Heat
Microwave
irradiation
Good tissue morphology straightforward

to perform (Cuevas et al., 1994) especially
for diagnostic purposes.
Detachment of section from slides, variability

in the reproducibility of immunostaining for the
use of different ovens (Cuevas et al., 1994)
Morphological details impairing by excessive heat
(Hunt et al., 1996; Chu et al., 2005)
In severe heating condition, nonspecic
background and false-positive stainings
increased (Ezaki et al., 1995; Takada et al., 2000)
Free-oating section damaging
(Jiao et al., 1999)
Pressure cooking
Speed of treatment, reproducibility of results

with large batches of slides, tested with single
or small number of primary antibodies, economy
of time and equipment costs (Norton et al., 1994)
Heat and digestion combination

Protein digestion
Non-specic endogenous peroxidase

activity reduced (Ezaki et al., 1995)
Ultrasound
Used at high-frequency, minimization

of tissue damage (Chu et al., 2005)
Etching (removal of resin)
(m-)ethoxide
Bleaching (reoxidation to osmium Na metaperiodate

tetroxide of reduced osmium)
Detergent
SDS
In some circumstances, the labeling efciency

is higher than in polar medium sections
(Brorson, 1998)
Tissue morphology often impaired (Cuevas et al.,

1994)
Severe tissue damage (Ordonez et al., 1988; Pileri
et al., 1997; Takada et al., 2000)
Possible epitope destruction (Pileri et al., 1997;
Van Hecke, 2002)
Increased background staining (Ezaki, 2000)
Unsuccessful and unreproducible results
(Hammoud and Van Norden, 2000; Leong
et al., 2002)
Loss of ultrastructural detail, section damage
(Stirling and Graff, 1995)
Low specicity (Silver et al., 1993; Yano et al., 2003)
Time-dependent section damage (Merighi, 1992)
Minimization of section contamination,

no wrinkled sections, unnecessary lm
suDDortina (Rajamannan et al., 2002)
Methods (2008), doi:10.1016/j.jim.2008.11.007
ARTICLE IN PRESS
In conclusion, the most important aim of each laboratory

should be to nd that protocol which gives the strongest
intensity of immunostaining, regardless of type and/or length
of xation.
As already mentioned, immunohistochemistry results are
also inuenced by other factors including the quantitative and
qualitative characteristics of antigen, afnity and concentrations of the antibody, sensitivity of the detection system
(Evers and Uylings, 1994; Pileri et al., 1997). For a detailed
discussion of these factors, the reader is referred to Goldstein
et al. (2007).
7. Conclusion
From the above cited examples of application, the following
important considerations can be drawn: a) antigen retrieval
can be obtained by different methods. Sometimes a combination of two or more methods is necessary; b) the appropriate
choice of the method reects the dependence of this one on
several factors, including type of xative and length of xation,
antigen and/or antibody nature; c) antigen retrieval is not
free from limitation and drawbacks, such as background
staining, false positive or negative, unmasking of endogenous
biotin. Although each retrieval approach can be useful to
unmask a given epitope, at the same time it can show a
disadvantage. Some advantages and disadvantages for different antigen retrieval approaches are summarised in Table 4.
Important factors, such as the delay in xation, xative
nature and its duration, as well as inappropriate applications
of antigen retrieval methods, can cause intra- and interlaboratory variability and reproducibility. Despite several
efforts, standardization in immunohistochemistry remains a
challenge. Thus, the use of standardized protocols which can
verify the methodological effectiveness for each antigen and
antibody under study is important . A wider application of
internal and external quality programs would improve greatly
the result accuracy and patient care.
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Journal of Immunological Methods xxx (2008) xxxxxx

Contents lists available at ScienceDirect

Journal of Immunological Methods

State of the art in antigen retrieval for immunohistochemistry

Corresponding author. Tel./fax: +39 095312017.

F. D'Amico et al. / Journal of Immunological Methods xxx (2008) xxxxxx

Most of them are synonyms.

which, other than economic advantage, lowers the possibility

Proteinase K, trypsin, chymotrypsin,

Source: microwave, autoclave, pressure

Glutaraldheyde shows a similar mechanism of action.

and ionic forces. On the contrary, at acidic or basic pH, the

Fig. 1. Simplied scheme of formaldehyde reaction with proteins.

F. D'Amico et al. / Journal of Immunological Methods xxx (2008) xxxxxx

structures with calcium ions and concluded that calcium

able to render the proteins susceptible to the unmasking effect

Retrieval agent Author(s)

Disruption of links between Heat

Shi et al., 1991; Suurmeijer

Suurmeijer and Boon, 1993a;

In Table 3 the most common theories on mechanisms of

cause a signicant problem with loss of antigenicity (Cattoretti

F. D'Amico et al. / Journal of Immunological Methods xxx (2008) xxxxxx

is reversible and caused by formaldehyde. Subsequently, the

Regarding calcium ions, their presence may inuence the

An increased background can be also obtained with the use

In general, dewaxed and rehydrated sections are treated with

F. D'Amico et al. / Journal of Immunological Methods xxx (2008) xxxxxx

and Rhodes, 2001, for reviews). Introduced in the early 1990s

parafn sections, treated with this method, exhibited similar

Antigen retrieval was carried out simply by immersing

TrisHCl, pH 9.0, at 95 C for 15 min (Evers and Uylings, 1994,

F. D'Amico et al. / Journal of Immunological Methods xxx (2008) xxxxxx

media, represent additional impairing factors for immunocytochemical reactions.

were Tris at 65 C overnight. Alternatively, the use of a

(Mayor et al., 1961; Berkowitz et al., 1968; Grube and

5.6. Minor approaches

F. D'Amico et al. / Journal of Immunological Methods xxx (2008) xxxxxx

considerably increased the reproducibility and consistency of

Although a successful antigen retrieval protocol depends

Good tissue morphology straightforward

Detachment of section from slides, variability

Speed of treatment, reproducibility of results

Heat and digestion combination

Non-specic endogenous peroxidase

Used at high-frequency, minimization

Etching (removal of resin)

Bleaching (reoxidation to osmium Na metaperiodate

In some circumstances, the labeling efciency

Tissue morphology often impaired (Cuevas et al.,

Minimization of section contamination,

In conclusion, the most important aim of each laboratory

F. D'Amico et al. / Journal of Immunological Methods xxx (2008) xxxxxx

Causton, B.E., 1984. The choice of resins for electron immunocytochemistry.

F. D'Amico et al. / Journal of Immunological Methods xxx (2008) xxxxxx

Newman, G.R., 1989. LR-White embedding medium for colloidal gold

F. D'Amico et al. / Journal of Immunological Methods xxx (2008) xxxxxx

polymer detection systems in immunohistochemistry. Arch. Pathol. Lab.

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