X-Message-Number: 4805
Date: Tue, 22 Aug 1995 19:48:43 +0200 (MET DST)
From: Eugene Leitl <>
Subject: fixation

=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=

Also in context of concurrent aldehyde/polyol perfusion:

"Avanced Organic Chemistry - Reactions, Mechanisms and
Structure", Jerry March, 3rd ed., John Wiley & Sons (1985), pp
789-791.

6-6 The Addition of Alcohols to Aldehyds and Ketones
(Dialkoxy-de-oxo-bisubstitution)

[picture]

Acetals and ketals are formed by treatment of aldehydes and
ketones, respectively, with alcohols in the presence of acid
catalysts. This is a reversible reaction, and acetal and ketals
can be hydrolyzed by treatment with acid (0-7). With small
						^^^^^^^^^^
unbranched aldehydes the equilibrium lies to the right. If it
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ [my
emphasis] is desired to prepare ketals, or acetals of larger
molecules, the equilibrium must be shifted, usually by removal
of water. This can be done by azeotropic destillation, ordinary
destillation, or the uses of a drying agent such as Al_2O_3 or a
molecular sieve. The reaction in neither direction is catalyzed
by bases, so most acetals and ketals are quite stable to bases,
though they are easily hydrolyzed by acids. This makes this
reaction a useful method of protection of aldehyde or ketone
functions from attack by bases. The reaction is of wide scope.
Most aldehydes are easily converted to acetals. With ketones the
process is more difficult, presumably by steric reasons, and the
reaction often fails, though many ketals, especially from cyclic
ketones, have been made in this manner. Many functional groups
can be presented without being affected. 1,2-Glycols and
1,3-glycols form cyclic acetals and ketals, e.g. [picture] and
these are often used to protect aldehydes and ketones.

[ Especially crosslinking agents' (as methanal) reactivity is
vastly enhanced due to occurence intermolecular reactions
(entropy favoured) with the polyol :( -- Eugene ]

=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=

"Techniques in Immunocytochemistry", Vol 1., Gillian R. Bullock,
Peter Petrusz, Edit., Academic Press, Inc. (London) Ltd.
(1982).

"Tissue Preparation Methods for Immunochemistry", Per
Brantzaeg, pp. 2-75.

"The Protein A-Gold (pAg) Technique - A Qualitative and
Quantitative Approach for Antigen Localization on Thin
Sections", J"urgen Roth, pp. 108-133.

"Light Microscopic Immunocytochemistry with Fixed, Unembedded
Tissues", Reinhard Grzanna, pp. 183-204.

=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=


Tissue Preparation Methods for Immunochemistry.

I. Introduction

[...]

The purpose of fixation is, in immunochemical terms, to arrest
enzymatic activity sufficiently rapidly to avoid structural
decomposition, to hinder diffusion of peptides and proteins
into and out of cells, and to fortify the tissue against
deletorious effects during the various stages in the
preparation of sections. Artifacts may develop when the tissue
specimens are subjected to dehydration, clearing and embedding,
and when the sections are being cut, floated, stretched, dried,
dewaxed, rinsed, incubated, and washed. In addition, structural
decomposition and diffusion artefacts my develop before the
fixation process takes place - either in vivo because of
denaturation or necrosis of cells, or in vitro because of
autolysis, osmotic damage, drying, or rough mechanical
treatment.

[...]

II. Principles of Tissue Preparation

A. Fixation Methods

Although fixation is necessary to avoid artefactual difussion of
soluble tissue components and decomposition of structures, it
constitutes in itself a major artefact since the living cell and
surroundings are fluid or semi-fluid in nature. A detailed
description of different fixatives and their action on various
tissue components can be found in several major texts on
histochemistry and hisopathology (Culling, 1974; Lillie and
Fullmer, 1976; Nairn, 1976; Pearse 1980).

Fixatives such as ethanol and methanol immobilize proteins and
carbohydrates by precipitation. The denaturing effect of these
fixatives is relatively mild and to large extent reversible.
Thus, proteins may be redissolved in a fairly native state after
ethanol fixation. It follows that adequate immobilization of
antigens in tissue sections is far from guaranteed. Moreover,
dehydration takes place simultaneously with the fixation process
so that morphological preservation may be unsatisfactory due to
shrinkage.

Immobilization of peptides and proteins is best afforded by
bifunctional cross-linking fixatives such as formaldehyded and
glutaraldehyde, which also preserve more adequately the
structures of cells and tissue. However, cross-linking
necessarily leads to more severe antigen denaturation than
precipitation; this particularly so for large protein antigenes
whose reactivity does not depend on primary structure alone but
also on conformational features (Kauzmann, 1959). Even the
antigenicity of peptides is adversely affected by the
aldehyde-based fixatives which react with primary amino groups,
and several alternative cross-linking agents have been suggested
for the localization of peptide hormones. These fixatives
include water-soluble carbodiimide (Kendall et al., 1971) and
the bifunctional reagent parabenzoquinone (Pearse and Polak,
1975).

Several variables will influence the effect of cross-linking
fixatives on antigenicity. Thus, when formaldehyde is used the
number of methylene bridges formed depends not only on the
concentrations of the fixative, but also on the temperature, pH
and time of exposure. The deletorious effect on antigen
reactivity may be partially reversed before tissue eembedding by
extensive washing in water or treeatment with sucrose (Eidelman
and Berschauer, 1969; Deng and Beutner, 1974).

The aldehyde-based fixatives induce both intermolecular and
intramolecular bridges. Due to such extensive formation of
cross-linkage formaldehyde, and even more so glutaraldehyde,
may in addition to denaturation may cause masking of antigens
by steric hindrance. This phenomenon is pronounced when the
actual antigen is mixed with high concentrations of other
proteins (Rognum et al., 1980; Hed and Enestrom, 1981). The
suggestions made by some authors (Sternberger, 1979) that the
deletorious effects of aldehyde-based fixatives can be
compensated for by use of a highly sensitive
immunohistochemical method is thus only partially true. It is
necessary to take into account that an uneven antigen masking
takes place according to location; interpretation of any
observed antigen distribution can only be meaningful if this
fact is kept in mind, regardless of whether immunofluorescence
or immunoenzyme methods are used for detection.

B. Exposure of Hidden Antigens [ various immunostain
enhancement procedures ]

C. Unmasking of Antigens Concealed During Fixation [ using
proteolytic digestion ]

D. Removal of Diffusible Proteins Before Fixation [ discerning
diffusible from membrane-bound/aggregated antigens ]

E. Pre-fixation Diffusion Artefacts

A problem that has received little attention, both in
immunobiological and immunopathological studies, is the fact
that immunoglobulins and complement factor C3 are present in
interstital fluid in relatively high concentrations and may,
therefore, enter cells by passive diffusion (Mason and
Biberfeld, 1980; Mason et al., 1980; Brandtzaeg, 1981b). Cells
subjected to such uptake often appear morphologically intact;
only slight plasma membrane damage may thus have been induced
prior to fixation, either in vivo or in vitro. Rough treatment
of the tissue specimen and retarded fixation contribute to this
aftefact. Kent (1966, 1967) showed that uptake of plasma
proteins in ischaemic myocardium occured according to the size
of the molecules and the degree of injury. In squamous
epithelia superficial degenerating cells commonly contain
plasma proteins in quantitaive proportions corresponding to the
content found in the underlying connective tissue (Brandtzaeg
and Kraus, 1965; Brandtzaeeg, 1975; Brandtzaeg et al. 1978).
Leakage of plasma proteins into columnar epithelium has
likewise been described (Mason and Piris, 1980). [...]

F. Post-fixation Diffusion Artefacts

It was pointed out above (Section II.A) that even large protein
anntigens may not always be sufficiently immobilized by ethanol
fixation to avoid their dissolution during incubation and
washing of the tissue section. We encountered this artifact in
immunofluorscence staining experiments when we prolonged the
incubation with fluorochrome conjugates from 30 min 20 h
(Brandtzaeg, 1981b). A decreased staining intensity could bee
ascribed both to loss of antigen and to partial neutralization
of the conjugate during the prolonged incubation time,
especially in the central areas of the section. This problem may
be even greater when working with unfixed or lighly fixed
cryostat sections. [...]

G. Conclusion

Ethanol acts as a fixative by protein precipitation whereas
formaldehyde immobilizes the proteins by the formation of
cross-linkages (Fig. 19). Immobilization of small antigens,
such as peptide hormones, can only be obtained safely with some
kind of cross-bridging fixative, and their antigenicity is
relatively well preserved by this procedure. The antigenicity
of protein antigens, an the other hand, does not depend on the
primary structure alone but also on conformational features,
which may be severely altered by the fixation process.

Due to extensive formation of cross-linkages, aldehyde-based
fixatives produce and additional artifact, namely masking of
antigens - particularly those mixed with high concentrations of
other proteins. [...]

III. Immunochemical Testing of Fixatives

A. Artificial Test Substrates [...]

B. Performance Testing on Biological Substrates [...]

1. Routine Formalin Fixation [...]

2. Glutaraldehyde-Formaldehyde

Glutaraldehyde is a dialdehyde and has a much greater
cross-linking potential than formaldehyde. Our tests with
artificial substrates showed that fixation with a combination
of 1 percent glutaraldehyde and 3.5 percent formaldehyde
yielded somewhat reduced detection sensivity for IgG comparted
with routine formalin fixation, and the masking was even more
pronounced at high antigen concentraions (Fig. 23). Despite
this properties, glutaraldehyde-containing fixatives have been
preferred in several studies based on PAP staining
(Sternberger, 1979). A significant advantage of the
glutaraldehyde-formaldehyde combination is that it gives
satisfactory preservation of tissue both for routine
histological staining methods and for electron microscopical
studies (McDowell and Trump, 1976). [...]

3. Baker's Formol Calcium

This fixative has been recommended for the preservation of
phospholipids in tissues. Our results with fixation of IgG and
IgA in artificial substrates indicated that Baker's formol
calcium yielded somewhat better detection sensitivity and much
less cross-bridging of protein than the other aldehyde-based
fixatives (Fig. 23). [...]

4. Formol Sublimate

[ several ugly mercuric chloride + additives preparations ]

5. Acetic Acid-Formol Saline

[...] Acetic acid-formol saline thus seems to be the
aldehyde-based fixative of choice for intracellular protein
antigens when proteolytic digestion of tissue section is
undesirable. However, this fixative does not permit the study
of extracellular or membrane-associated antigens. The results
of Curran and Gregory (1980) indicated that it is the low pH of
the formaldehyde solution rather than the acetic acid per se
that explains the favourable results.

6. Bouin's Fluid

This fixative is known to penetrate rapidly and to cause little
shrinkage. In addition to formaldehyde and acetic acid, it
contains picric acid, which precipitates proteins and combines
with them to form picrates and also produces intermolecular
salt links (Pearse, 1980). [...]

7. Susa Fixative

This fixative has properties similiar tot Bouin's fluid, but
contains trichloroacetic acid and mercuric chloride instead of
picric acid. [...] Alltogether, there were no apparent
advantages in using Susa fixative for immunochemistry of
immunoglobulins.

8. Carbodiimide

Water-soluble carbodiimides have been widely used to prepare
immunogenic conjugates by coupling small peptides to carrier
proteins. They effect polymerization by initial attack of
carboxyl groups to give an acylisourea, which next may react
with an adjacent amino group to crosslink through an amide bond
(Stark, 1970). A requirement for cross-linking, therefore, is
probably close proximity between carboxyl and amino groups;
this may explain that although carbodiimides are as efficient
as aldehydes for insolubilization of proteins (Yamamoto and
Yasuda, 1977), we found substantially less masking of
antigenicity after tissue fixation with the former (Brandtzaeg
and Rognum, 1982a,b). [...] Carbodiimide is thus a
cross-linking fixative that for protein antigens gives a result
similiar to that obtained after ethanol fixation (Fig. 23). It
has recently been shown that the cytosol matrix remains
permeable after fixation with carbodiimide even when 0.3
percent glutaraldehyde is addd to it (Willingham and Yamada,
1979). This combination enhances morphological preservation and
offers promises for application in immunoelectron microscopical
localization studies.

[ On the whole carbodiimides might be promising auxiliary
perfusion agents, particularly since they do not react with
polyols but require carboxyl/amine group for a crosslink. Of
course, their influence on perfusion (blood-brain barrier) has
to be checked first. Membrane-shuttle agents might be
instrumental here, albeit artefacting on their own rights
-- Eugene ]

9. Ethanol

The Sainte-Marie (1962) cold ethanol-fixation procedure
afforded superior detection sensitivity for IgG and IgA in
artificial substrates, and superior immunofluorescence staining
intensity for epithelial SC and IgA (Fig. 23). The same held
true for immunoglobulin-producing cells of all isotypes when
the prefixation washing step was included (Section II. D), and
for intracellular J chain when the sections were treated with
acid urea (Section II. B). The advantages and drawbacks of
tissue preparation methods based on ethanol fixation have been
further discussed in Sections II. C, II. E, II.F and II.G and
in previous publications (Brandtzaeg, 1974, 1981b).

10. Conclusion

[ not much new, here ]

IV. Choice of Tissue Preparation Method

A. Extracellular and Basement Membrane-Assotiated Protein
Antigens [...]

B. Cell-Surface and Cytoplasmic Immunoglobulin Components
Including J Chain and SC [...]

C. Cytoplasmic Enzymes [...]

D. Cytoplasmic Hormones [...]

E. Miscellaneous Cellular and Tissue Antigens [...]

F. Conclusion

Tissue preparation is the cornerstone of immunohistochemistry,
but is still a subject of much antiquity and ambiguity. There
is a need to clarify basic mechanisms rather than to introduce
unjustified modifications of existing methods. Furthere
progress can only be made when knowledge of the various
antigens and their microenvironment is considered together with
knowledge of the chemistry of fixation.

Statements made in the literature about a general applicability
of certain fixatives are likely to be false. It seems that many
antigens require a tailor-made tisue preparation technique for
optimal preservation and precise localization. From a
morphological point of view cross-linking fixatives are
generally preferable, but the antigenic masking caused by them
is, apparently, a more universal problem in
immunohistochemistry than earlier believed. Although
proteolytic unmasking to some extent is possible, optimal
preservation of antigenicity and morphology is not always
compatible with such tretment.

It is important that those entering the field of
immunohistochemistry be aware of the fact that success is not a
matter of mere stain technology in terms of antigen labeling by
immunological reactions. The primary and crucial substrate for
this reactions is the antigenicity that has been preserved and
rendered accessible in the tissue section. In addition comes
the goal of adequate morphology.

V. Acknowledgements [...]

VI. References [...]

=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=

"The Protein A-Gold (pAg) Technique - A Qualitative and
Quantitative Approach for Antigen Localization on Thin
Sections", J"urgen Roth, pp. 108-133.

[...]

A. Tissue Fixation

In order to obtain adequate immunocytochemical localization of
antigens, the fixatives used should stabilize cellular
structures to such an extent that artefactual diffusion and
replacement of antigenic material or its extraction is
prevented and good preservation of cellular fine structure is
achieved. On the other hand, the fixative should preserve as
much as possible the tertiary structure of antigens to retain a
good reactivity with the antibodies. A common phenomenon in
post-embedding staining procedures is that excellent structural
preservation often results in drastic diminution of
antigenicity since fixatives affect fine structure and
antigenicity inversely. Therefore, conditions of fixation have
to be devised which preserve adequately both cellular fine
structure and antigenicity. Among the fixatives used in
electron microscopy, aldehydes fulfil these requirements most
closely. Other fixatives, such as carboddimide (Polak et al.,
1972; Hassel and Hand, 1974; Yamamoto and Yasuda, 1977) and
diimidoesters (McLean and Singer, 1970; Ono et al., 1976) have
been proposed, but have not been widely tested as yet.
Kraehenbuhl et al. (1977) have shown that individual enzymes
are affected differently by glutaraldehyde but that low
concentrations of glutaraldehyde (0.5 percent) are commensurate
with good preservation of both the antigenic properties of
pancreatic enzymes and the fine structure of pancreatic tissue.
For this reason, we routinely use a 2 h fixation with 0.5
percent glutaraldehyde solution in PBS at room temperature for
pancreatic tissue (Bendayan et al., 1980). We also tested
fixation with picric acid-formaldehyde solution (Stefanini et
al., 1967) which gave a more intense labelling compared to
glutaraldehyde fixation but at the expense of fine structural
preservation. In our studies on localization of polypeptide
hormones we found good preservation of antigenicity after
fixation with relatively high concentration of aldehyde (up to
4 percent glutaraldehyde concentration or mixtures of 3 percent
glutaraldehyde with 2 percent formaldehyde). Osmium fixation
severely influences the antigenicity of many proteins. Only a
few antigens have been shown to resist such a fixation protocol
(Nakane, 1971; Erlandsen et al., 1979). We observed that
aldehyde fixation followed by 1 percent osmium tetroxide
fixation for 1 h allowed localization of insulin in pancreatic
B cells as in the only aldehyde fixed tissue. On the contrary,
we found that aldehyde fixation followed by 1 percent osmium
tetroxide for 15 min reduced the labelling intensity for
amylase in rat pancreatic tissue by 70 percent.

In general, localization of an antigen should be tried first on
tissue fixed with 0.5 to 1 percent buffered glutaraldehyde
solution since this fixative sufficiently preserves many
antigens for subsequent demonstration with the pAg technique.
However, for each particular cell type or tissue one has to
determine the fixation conditions which are subject: fixation

=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=

Also in context of concurrent aldehyde/polyol perfusion:

"Avanced Organic Chemistry - Reactions, Mechanisms and
Structure", Jerry March, 3rd ed., John Wiley & Sons (1985), pp
789-791.

6-6 The Addition of Alcohols to Aldehyds and Ketones
(Dialkoxy-de-oxo-bisubstitution)

[picture]

Acetals and ketals are formed by treatment of aldehydes and
ketones, respectively, with alcohols in the presence of acid
catalysts. This is a reversible reaction, and acetal and ketals
can be hydrolyzed by treatment with acid (0-7). With small
						^^^^^^^^^^
unbranched aldehydes the equilibrium lies to the right. If it
^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ [my
emphasis] is desired to prepare ketals, or acetals of larger
molecules, the equilibrium must be shifted, usually by removal
of water. This can be done by azeotropic destillation, ordinary
destillation, or the uses of a drying agent such as Al_2O_3 or a
molecular sieve. The reaction in neither direction is catalyzed
by bases, so most acetals and ketals are quite stable to bases,
though they are easily hydrolyzed by acids. This makes this
reaction a useful method of protection of aldehyde or ketone
functions from attack by bases. The reaction is of wide scope.
Most aldehydes are easily converted to acetals. With ketones the
process is more difficult, presumably by steric reasons, and the
reaction often fails, though many ketals, especially from cyclic
ketones, have been made in this manner. Many functional groups
can be presented without being affected. 1,2-Glycols and
1,3-glycols form cyclic acetals and ketals, e.g. [picture] and
these are often used to protect aldehydes and ketones.

[ Especially crosslinking agents' (as methanal) reactivity is
vastly enhanced due to occurence intermolecular reactions
(entropy favoured) with the polyol :( -- Eugene ]

=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=

"Techniques in Immunocytochemistry", Vol 1., Gillian R. Bullock,
Peter Petrusz, Edit., Academic Press, Inc. (London) Ltd.
(1982).

"Tissue Preparation Methods for Immunochemistry", Per
Brantzaeg, pp. 2-75.

"The Protein A-Gold (pAg) Technique - A Qualitative and
Quantitative Approach for Antigen Localization on Thin
Sections", J"urgen Roth, pp. 108-133.

"Light Microscopic Immunocytochemistry with Fixed, Unembedded
Tissues", Reinhard Grzanna, pp. 183-204.

=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=


Tissue Preparation Methods for Immunochemistry.

I. Introduction

[...]

The purpose of fixation is, in immunochemical terms, to arrest
enzymatic activity sufficiently rapidly to avoid structural
decomposition, to hinder diffusion of peptides and proteins
into and out of cells, and to fortify the tissue against
deletorious effects during the various stages in the
preparation of sections. Artifacts may develop when the tissue
specimens are subjected to dehydration, clearing and embedding,
and when the sections are being cut, floated, stretched, dried,
dewaxed, rinsed, incubated, and washed. In addition, structural
decomposition and diffusion artefacts my develop before the
fixation process takes place - either in vivo because of
denaturation or necrosis of cells, or in vitro because of
autolysis, osmotic damage, drying, or rough mechanical
treatment.

[...]

II. Principles of Tissue Preparation

A. Fixation Methods

Although fixation is necessary to avoid artefactual difussion of
soluble tissue components and decomposition of structures, it
constitutes in itself a major artefact since the living cell and
surroundings are fluid or semi-fluid in nature. A detailed
description of different fixatives and their action on various
tissue components can be found in several major texts on
histochemistry and hisopathology (Culling, 1974; Lillie and
Fullmer, 1976; Nairn, 1976; Pearse 1980).

Fixatives such as ethanol and methanol immobilize proteins and
carbohydrates by precipitation. The denaturing effect of these
fixatives is relatively mild and to large extent reversible.
Thus, proteins may be redissolved in a fairly native state after
ethanol fixation. It follows that adequate immobilization of
antigens in tissue sections is far from guaranteed. Moreover,
dehydration takes place simultaneously with the fixation process
so that morphological preservation may be unsatisfactory due to
shrinkage.

Immobilization of peptides and proteins is best afforded by
bifunctional cross-linking fixatives such as formaldehyded and
glutaraldehyde, which also preserve more adequately the
structures of cells and tissue. However, cross-linking
necessarily leads to more severe antigen denaturation than
precipitation; this particularly so for large protein antigenes
whose reactivity does not depend on primary structure alone but
also on conformational features (Kauzmann, 1959). Even the
antigenicity of peptides is adversely affected by the
aldehyde-based fixatives which react with primary amino groups,
and several alternative cross-linking agents have been suggested
for the localization of peptide hormones. These fixatives
include water-soluble carbodiimide (Kendall et al., 1971) and
the bifunctional reagent parabenzoquinone (Pearse and Polak,
1975).

Several variables will influence the effect of cross-linking
fixatives on antigenicity. Thus, when formaldehyde is used the
number of methylene bridges formed depends not only on the
concentrations of the fixative, but also on the temperature, pH
and time of exposure. The deletorious effect on antigen
reactivity may be partially reversed before tissue eembedding by
extensive washing in water or treeatment with sucrose (Eidelman
and Berschauer, 1969; Deng and Beutner, 1974).

The aldehyde-based fixatives induce both intermolecular and
intramolecular bridges. Due to such extensive formation of
cross-linkage formaldehyde, and even more so glutaraldehyde,
may in addition to denaturation may cause masking of antigens
by steric hindrance. This phenomenon is pronounced when the
actual antigen is mixed with high concentrations of other
proteins (Rognum et al., 1980; Hed and Enestrom, 1981). The
suggestions made by some authors (Sternberger, 1979) that the
deletorious effects of aldehyde-based fixatives can be
compensated for by use of a highly sensitive
immunohistochemical method is thus only partially true. It is
necessary to take into account that an uneven antigen masking
takes place according to location; interpretation of any
observed antigen distribution can only be meaningful if this
fact is kept in mind, regardless of whether immunofluorescence
or immunoenzyme methods are used for detection.

B. Exposure of Hidden Antigens [ various immunostain
enhancement procedures ]

C. Unmasking of Antigens Concealed During Fixation [ using
proteolytic digestion ]

D. Removal of Diffusible Proteins Before Fixation [ discerning
diffusible from membrane-bound/aggregated antigens ]

E. Pre-fixation Diffusion Artefacts

A problem that has received little attention, both in
immunobiological and immunopathological studies, is the fact
that immunoglobulins and complement factor C3 are present in
interstital fluid in relatively high concentrations and may,
therefore, enter cells by passive diffusion (Mason and
Biberfeld, 1980; Mason et al., 1980; Brandtzaeg, 1981b). Cells
subjected to such uptake often appear morphologically intact;
only slight plasma membrane damage may thus have been induced
prior to fixation, either in vivo or in vitro. Rough treatment
of the tissue specimen and retarded fixation contribute to this
aftefact. Kent (1966, 1967) showed that uptake of plasma
proteins in ischaemic myocardium occured according to the size
of the molecules and the degree of injury. In squamous
epithelia superficial degenerating cells commonly contain
plasma proteins in quantitaive proportions corresponding to the
content found in the underlying connective tissue (Brandtzaeg
and Kraus, 1965; Brandtzaeeg, 1975; Brandtzaeg et al. 1978).
Leakage of plasma proteins into columnar epithelium has
likewise been described (Mason and Piris, 1980). [...]

F. Post-fixation Diffusion Artefacts

It was pointed out above (Section II.A) that even large protein
anntigens may not always be sufficiently immobilized by ethanol
fixation to avoid their dissolution during incubation and
washing of the tissue section. We encountered this artifact in
immunofluorscence staining experiments when we prolonged the
incubation with fluorochrome conjugates from 30 min 20 h
(Brandtzaeg, 1981b). A decreased staining intensity could bee
ascribed both to loss of antigen and to partial neutralization
of the conjugate during the prolonged incubation time,
especially in the central areas of the section. This problem may
be even greater when working with unfixed or lighly fixed
cryostat sections. [...]

G. Conclusion

Ethanol acts as a fixative by protein precipitation whereas
formaldehyde immobilizes the proteins by the formation of
cross-linkages (Fig. 19). Immobilization of small antigens,
such as peptide hormones, can only be obtained safely with some
kind of cross-bridging fixative, and their antigenicity is
relatively well preserved by this procedure. The antigenicity
of protein antigens, an the other hand, does not depend on the
primary structure alone but also on conformational features,
which may be severely altered by the fixation process.

Due to extensive formation of cross-linkages, aldehyde-based
fixatives produce and additional artifact, namely masking of
antigens - particularly those mixed with high concentrations of
other proteins. [...]

III. Immunochemical Testing of Fixatives

A. Artificial Test Substrates [...]

B. Performance Testing on Biological Substrates [...]

1. Routine Formalin Fixation [...]

2. Glutaraldehyde-Formaldehyde

Glutaraldehyde is a dialdehyde and has a much greater
cross-linking potential than formaldehyde. Our tests with
artificial substrates showed that fixation with a combination
of 1 percent glutaraldehyde and 3.5 percent formaldehyde
yielded somewhat reduced detection sensivity for IgG comparted
with routine formalin fixation, and the masking was even more
pronounced at high antigen concentraions (Fig. 23). Despite
this properties, glutaraldehyde-containing fixatives have been
preferred in several studies based on PAP staining
(Sternberger, 1979). A significant advantage of the
glutaraldehyde-formaldehyde combination is that it gives
satisfactory preservation of tissue both for routine
histological staining methods and for electron microscopical
studies (McDowell and Trump, 1976). [...]

3. Baker's Formol Calcium

This fixative has been recommended for the preservation of
phospholipids in tissues. Our results with fixation of IgG and
IgA in artificial substrates indicated that Baker's formol
calcium yielded somewhat better detection sensitivity and much
less cross-bridging of protein than the other aldehyde-based
fixatives (Fig. 23). [...]

4. Formol Sublimate

[ several ugly mercuric chloride + additives preparations ]

5. Acetic Acid-Formol Saline

[...] Acetic acid-formol saline thus seems to be the
aldehyde-based fixative of choice for intracellular protein
antigens when proteolytic digestion of tissue section is
undesirable. However, this fixative does not permit the study
of extracellular or membrane-associated antigens. The results
of Curran and Gregory (1980) indicated that it is the low pH of
the formaldehyde solution rather than the acetic acid per se
that explains the favourable results.

6. Bouin's Fluid

This fixative is known to penetrate rapidly and to cause little
shrinkage. In addition to formaldehyde and acetic acid, it
contains picric acid, which precipitates proteins and combines
with them to form picrates and also produces intermolecular
salt links (Pearse, 1980). [...]

7. Susa Fixative

This fixative has properties similiar tot Bouin's fluid, but
contains trichloroacetic acid and mercuric chloride instead of
picric acid. [...] Alltogether, there were no apparent
advantages in using Susa fixative for immunochemistry of
immunoglobulins.

8. Carbodiimide

Water-soluble carbodiimides have been widely used to prepare
immunogenic conjugates by coupling small peptides to carrier
proteins. They effect polymerization by initial attack of
carboxyl groups to give an acylisourea, which next may react
with an adjacent amino group to crosslink through an amide bond
(Stark, 1970). A requirement for cross-linking, therefore, is
probably close proximity between carboxyl and amino groups;
this may explain that although carbodiimides are as efficient
as aldehydes for insolubilization of proteins (Yamamoto and
Yasuda, 1977), we found substantially less masking of
antigenicity after tissue fixation with the former (Brandtzaeg
and Rognum, 1982a,b). [...] Carbodiimide is thus a
cross-linking fixative that for protein antigens gives a result
similiar to that obtained after ethanol fixation (Fig. 23). It
has recently been shown that the cytosol matrix remains
permeable after fixation with carbodiimide even when 0.3
percent glutaraldehyde is addd to it (Willingham and Yamada,
1979). This combination enhances morphological preservation and
offers promises for application in immunoelectron microscopical
localization studies.

[ On the whole carbodiimides might be promising auxiliary
perfusion agents, particularly since they do not react with
polyols but require carboxyl/amine group for a crosslink. Of
course, their influence on perfusion (blood-brain barrier) has
to be checked first. Membrane-shuttle agents might be
instrumental here, albeit artefacting on their own rights
-- Eugene ]

9. Ethanol

The Sainte-Marie (1962) cold ethanol-fixation procedure
afforded superior detection sensitivity for IgG and IgA in
artificial substrates, and superior immunofluorescence staining
intensity for epithelial SC and IgA (Fig. 23). The same held
true for immunoglobulin-producing cells of all isotypes when
the prefixation washing step was included (Section II. D), and
for intracellular J chain when the sections were treated with
acid urea (Section II. B). The advantages and drawbacks of
tissue preparation methods based on ethanol fixation have been
further discussed in Sections II. C, II. E, II.F and II.G and
in previous publications (Brandtzaeg, 1974, 1981b).

10. Conclusion

[ not much new, here ]

IV. Choice of Tissue Preparation Method

A. Extracellular and Basement Membrane-Assotiated Protein
Antigens [...]

B. Cell-Surface and Cytoplasmic Immunoglobulin Components
Including J Chain and SC [...]

C. Cytoplasmic Enzymes [...]

D. Cytoplasmic Hormones [...]

E. Miscellaneous Cellular and Tissue Antigens [...]

F. Conclusion

Tissue preparation is the cornerstone of immunohistochemistry,
but is still a subject of much antiquity and ambiguity. There
is a need to clarify basic mechanisms rather than to introduce
unjustified modifications of existing methods. Furthere
progress can only be made when knowledge of the various
antigens and their microenvironment is considered together with
knowledge of the chemistry of fixation.

Statements made in the literature about a general applicability
of certain fixatives are likely to be false. It seems that many
antigens require a tailor-made tisue preparation technique for
optimal preservation and precise localization. From a
morphological point of view cross-linking fixatives are
generally preferable, but the antigenic masking caused by them
is, apparently, a more universal problem in
immunohistochemistry than earlier believed. Although
proteolytic unmasking to some extent is possible, optimal
preservation of antigenicity and morphology is not always
compatible with such tretment.

It is important that those entering the field of
immunohistochemistry be aware of the fact that success is not a
matter of mere stain technology in terms of antigen labeling by
immunological reactions. The primary and crucial substrate for
this reactions is the antigenicity that has been preserved and
rendered accessible in the tissue section. In addition comes
the goal of adequate morphology.

V. Acknowledgements [...]

VI. References [...]

=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=

"The Protein A-Gold (pAg) Technique - A Qualitative and
Quantitative Approach for Antigen Localization on Thin
Sections", J"urgen Roth, pp. 108-133.

[...]

A. Tissue Fixation

In order to obtain adequate immunocytochemical localization of
antigens, the fixatives used should stabilize cellular
structures to such an extent that artefactual diffusion and
replacement of antigenic material or its extraction is
prevented and good preservation of cellular fine structure is
achieved. On the other hand, the fixative should preserve as
much as possible the tertiary structure of antigens to retain a
good reactivity with the antibodies. A common phenomenon in
post-embedding staining procedures is that excellent structural
preservation often results in drastic diminution of
antigenicity since fixatives affect fine structure and
antigenicity inversely. Therefore, conditions of fixation have
to be devised which preserve adequately both cellular fine
structure and antigenicity. Among the fixatives used in
electron microscopy, aldehydes fulfil these requirements most
closely. Other fixatives, such as carboddimide (Polak et al.,
1972; Hassel and Hand, 1974; Yamamoto and Yasuda, 1977) and
diimidoesters (McLean and Singer, 1970; Ono et al., 1976) have
been proposed, but have not been widely tested as yet.
Kraehenbuhl et al. (1977) have shown that individual enzymes
are affected differently by glutaraldehyde but that low
concentrations of glutaraldehyde (0.5 percent) are commensurate
with good preservation of both the antigenic properties of
pancreatic enzymes and the fine structure of pancreatic tissue.
For this reason, we routinely use a 2 h fixation with 0.5
percent glutaraldehyde solution in PBS at room temperature for
pancreatic tissue (Bendayan et al., 1980). We also tested
fixation with picric acid-formaldehyde solution (Stefanini et
al., 1967) which gave a more intense labelling compared to
glutaraldehyde fixation but at the expense of fine structural
preservation. In our studies on localization of polypeptide
hormones we found good preservation of antigenicity after
fixation with relatively high concentration of aldehyde (up to
4 percent glutaraldehyde concentration or mixtures of 3 percent
glutaraldehyde with 2 percent formaldehyde). Osmium fixation
severely influences the antigenicity of many proteins. Only a
few antigens have been shown to resist such a fixation protocol
(Nakane, 1971; Erlandsen et al., 1979). We observed that
aldehyde fixation followed by 1 percent osmium tetroxide
fixation for 1 h allowed localization of insulin in pancreatic
B cells as in the only aldehyde fixed tissue. On the contrary,
we found that aldehyde fixation followed by 1 percent osmium
tetroxide for 15 min reduced the labelling intensity for
amylase in rat pancreatic tissue by 70 percent.

In general, localization of an antigen should be tried first on
tissue fixed with 0.5 to 1 percent buffered glutaraldehyde
solution since this fixative sufficiently preserves many
antigens for subsequent demonstration with the pAg technique.
However, for each particular cell type or tissue one has to
determine the fixation conditions which are compatible with
adequate preservation of fine structure and antigenicity.

=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=


Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=4805