Fixation of biological specimens

WOLF D. KUHLMANN, M.D.

Division of Radiooncology, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany

Fixation is usually the first step to prepare biological specimens for microscopy. Any
treatment which will preserve cell structure and its biochemical composition can be deemed
to be fixation. Of course, the quality of fixation is the key for all following steps which are
necessary in histological research. Hence, preservation of cells with minimal alteration of
morphology and virtually no loss of molecules is essential in tissue preservation. In this sense,
tissue fixation has to preserve the cells in as life-like a state as possible. Moreover, fixation
should protect biological specimens from the denaturing effects of dehydration and all further
processings.
Historically, the majority of fixative solutions and fixation procedures were developed for
light microscopic studies. The choice of a fixation protocol will largely depend on the
analyses to be performed. All processes of tissue preparation should be as reproducible as
possible. Obviously, there exists no ideal fixative for all study types. It can be even necessary
to apply different fixation protocols for different structural elements within a given tissue.
Organic and inorganic reagents have been used successfully for tissue stabilisation and a
number of histological stainings. One of the most commonly used fixatives in histology is still
formalin, i.e. formaldehyde dissolved in aqueous solutions (FOX CH et al., 1985; KIERNAN JA,
2008).
With the invention of the electron microscope it was soon realized that the usual fixation
schedules developed for light microscopy are not adequate for ultrastructural studies. With
regard to high resolution morphology, electron microscopy is going to be more exacting than
light microscopy. Common histopathological fixatives and tissue preparations allow not to
reveal ultrastructural details satisfactorily because of inherent artefacts such as shrinkage,
extraction and precipitation phenomena.
In the search for adequate fixatives for electron microscopy, major advances were made with
the introduction of glutaraldehyde as a primary fixative which is followed by osmium
tetroxide as a secondary fixative (SABATINI DD et al., 1963; SABATINI DD et al., 1964). The
convenience of this double fixation and the excellent preservation of fine structure has made
this procedure to be the standard in biological electron microscopy. The success of
glutaraldehyde, however, led to the reexamination of other aldehydes as primary fixatives. It
became obvious that f.e. formaldeyhde, freshly prepared from paraformaldehyde (ROBERTSON
JD et al., 1963), compares favourably with glutaraldehyde. Furthermore, mixtures of both
aldehydes and were found to be superior to either of the aldehydes used alone (KARNOVSKY
MJ, 1965).
Histological fixatives contain one or more reactive components. It can be expected that no
matter what reagent is used the molecular structures may become altered: either chemically
by reaction with molecules (e.g. cross-linking of proteins) or physically by precipitation. Both
actions can result in denaturation. Likewise, structures and biomolecules will be disrupted or
being lost during fixation (as well as afterwards during subsequent tissue processing). We

know by experience that fixation methods which offer good morphological preservation are
prone to pitfalls in cytochemical methods such as enzyme histochemistry or antigen-antibody
reactions in immunohistology. For this reason, cell and tissue fixation must be adapted to the
special needs. This is usually done by a number of defined trials to monitor morphological
integrity and molecular reactivity likewise.

Fixatives and some impacts of fixation

Many fixation types exist for various purposes. Fixatives which are chosen for anatomical
studies (whole tissue structure) are often different from those for cytological purposes (cell
smears, cell suspensions from body fluids or prepared from tissues). Apart from such aspects,
fixation has to reconcile with histochemical features of the studied material (enzymes,
antigens etc.). This discrimination is not exclusive because fixatives can be satisfactory for
different purposes.
Fixatives which combine with proteins are called additive fixatives, and those which
precipitate proteins are coagulant or precipitant fixatives. On the basis of this categorization,
fixatives may be roughly classified into the following groups.

Additive fixatives, e.g. aldehydes, potassium dichromate, osmium tetroxide.

Coagulant fixatives, e.g. acetone, ethanol,methanol, trichloroacetic acid.

Additive coagulant fixatives, e.g. chromic acid, mercuric chloride, picric acid.

Non-additive non-coagulant fixatives, e.g. acetic acid.

A great number of factors will influence the fixation process. Many variables must be
cinsidered and controlled.

Time interval from removal of tissue to fixation: fixation should be started as fast as
possible. Also, avoid drying of tissue between the steps of tissue removal and fixation.

pH, buffering capacity and osmolality: fixation of animal cells is best carried out near
neutral pH. Hypertonic fixatives will lead to shrinkage, and hypotonic solutions result in
cell swelling.

Penetrability of fixative: this depends on the diffusibility of the individual components

and the size of the tissue specimens.

Volume: a high ratio of fixative to tissue will ensure good fixation process. In this sense,
it is best to change the fixative solution several times during the fixation process.

Temperature: fixation is mainly a chemical process, thus, increasing the temperature will
increase the fixation speed.

Concentration: too low and too high a concentration will have adverse effects, and
artefacts can be produced.
Vehicles and additives: some salts can have denaturing effects while others such as
ammonium sulfate can stabilise proteins. Tannic acid can be useful because it penetrates
tissues easily and precipitates polypeptides and proteins. Phenol has an accelerating effect
on formaldehyde fixation. Transitional metal salts such as zinc sulfate helps in the
formation of insoluble protein and polypeptide complexes, thus enhancing antigen
preservation. It is possible to supplement fixatives with detergents with the aim to
enhance subsequent microtechniques. It must be noted, however, that this type of primary
fixation is limited to special applications because detergents in general have deleterious
effects on cellular details.

Duration of fixation: the optimal fixation time depends on multiple factors, f.e. on the
thickness of the tissue specimen and, of course, most of the above mentioned features of
the fixation process (temperature, buffering capacity, penetrability of the fixative
substances, volume ratios). Apart from loss of antigenic reactivity, prolonged fixation will
cause shrinkage and hardening of specimens. In certain cases, degradations can be also
expected.
The influence of different fixation methods such as osmolality, ionic strength etc. on the fine
structure and molecules of cells was shown impressively in the electron microscope
(MAUNSBACH AB, 1966a, 1966b; DOGGENWEILER CF and HEUSER JE, 1967).

Fixation procedures
Fixation of biological specimens can be realized by two typical ways: (a) perfusion fixation
via bloodflow; and (b) immersion fixation by which tissue samples are immersed into the
fixative solution. It depends on the type of tissue which procedure is to favour. Alternatively,
phase partition fixation may be preferred under certain conditions (LEIST DP et al., 1986).
Fixation is in most cases done by immersion of specimens into the fixative solution. Tissue
specimens should be placed in fixative as rapidly as possible after they have been removed
from the body. The fixative solution should be at least 20 times the volume of the tissue.
Furthermore, the fixative should be changed several times; the time of fixation is dependent
on the thickness of the tissue.
Perfusion fixation is generally preferred for large organs or when the fine structure of an
organ is critically dependent on a continous blood supply. A number of perfusion methods
have been published and, most probably, many more unpublished variants will exist. A
helpful description of perfusion fixation, perfusion fluids and the route of injection is given by
AM GLAUERT (1975).
In the case of cell smears or single cells which are collected on glass slides by
cytocentrifugation, merely air-drying may also act as a form of preservation. The
requirements of further fixation vary according to the techniques needed for the visualization
of morphological details and the molecules (cell function) to be studied. Cells in culture
growing as monolayer are first rinsed with balanced salt solution, then treated with fixative
(by immersion fixation). The choice of the right fixative and the right buffer solution is a
matter of careful selection since monolayer cells are unprotected against osmotic changes as
compared with cells in tissues.
The handling of isolated cells or cell fractions is sometimes difficult. Therefore, numerous
methods were developed to collect the cells and to perform a primary fixation. Usually,
centrifugation steps are included (RYTER A and KELLENBERGER E, 1958; MALAMED S, 1963;
ANDERSON DR, 1965; GLAUERT AM and THORNLEY MJ, 1966; CHARRET R and FAURFREMIET E, 1967; MARIKOVSKY Y and DANON D, 1967; HIRSCH JG and FEDORKO M, 1968;
MCCOMBS RM et al., 1968; SHANDS JW, 1968; FURTADO JS, 1970; SAWICKI W and LIPETZ J,
1971; KUHLMANN WD and VIRON A, 1972).

Cells are fixed in suspension by adding adequate volumes of fixation solution, followed
by centrifugation in order to obtain a pellet. Pellets are washed and further treated
depending on the selected study protocol.

Cells are first pelleted by centrifugation. After removal of the supernatant, fixative is
carefully added to the pellet so as to keep the pellet intact; alternatively, the pelleted cells
can be resuspended within the added fixative. Washed pellets (or centrifugation steps for
cell washings) are submitted to selective study protocols.

Cell suspensions or cell pellets which tend to disintegrate upon further handling can be
encapsulated by several means such as fibrin clotting, suspension in agar gel, crosslinking with bovine serum albumin and others.
In some cases, primary fixation will not be sufficient for cellular preservation so that the
specimens (tissue blocks, cells in suspension or as monolayers) have to be submitted to
postfixation. Postfixation in osmium is very common preparative step in electron microscopy.
In this case, fixed cell preparations are transferred from the washing buffer into a postfixation
solution of buffered osmium tetroxide which is followed by dehydration and embedding.
For a number of reasons, chemical fixation should to be avoided at all so as not to disturb
cellular fine structures and their molecular compositions. Thus, possible alternatives were
searched for, and with the advances made in freezing techniques, vitrous ice was found as a
useful tool in ultrastructural research by which both chemical fixation and chemical
embedding matrix can be avoided (PLATTNER H and BACHMANN L, 1982).

Cellular components and the process of fixation

Unfortunately, fixation is often associated with variable loss of molecular properties (f.e.
antigenicity), or fixation will render molecular sites (tissue antigen) inaccessible. In order to
optimize tissue fixation, one has to implement a strict strategy of tissue sampling which
allows to control the nature and duration of fixation. This will at least help to compromise on
morphology and preservation of function (f.e. antigenicity). Since results of tissue are difficult
to predict, attempts of antigen retrieval are straightforward to render the desired antigen
accessible for immuno-staining.
Tissues are composed of both more and less soluble substances. Secretory products are part of
the soluble ones whereas membranes and other high molecular complexes including
components of the cytoskeleton are organized as relatively insoluble structures. Hence, it is
readily understood that tissue molecules not being bound to solid structures can easily diffuse
from their sites unless sufficient stabilization is performed by appropriate tissue sampling.
Fixation may break membrane structures. Consequently, molecules are allowed to penetrate
hitherto forbidden compartments or to escape from their original sites.
The stabilization step must be in consent with all subsequent stages of tissue preparation:
dehydration, embedment, microtomy and staining schedules. An ideal fixative is
characterized by rapid penetration into the specimens and compatibility with conventional
and molecular specific stains. Unfortunately, all these properties are difficult to find in a
single fixative. Different fixatives will give different degrees of fixation outcomes. Some of
them can be of advantage for routine stainings but at the same time of great disadvantage for
the more selective ones.
Fixation is a compromise and will vary according to the techniques applied. The requirements
of fixation for cytology are different from those for histopathology or for electron microscopy.
In special cases, fixation solutions must be designed for the selected application.

For most purposes, tissue fixation is done by chemical reactions. Two main types of fixatives
are regularly used in histology: cross-linking fixatives and coagulating fixatives. Especially
aldehydes and organic solvents are common. Fixation procedures based on water
replacement by substitution with inert molecules (e.g. ethylene glycol) and physical means
(freeze-drying, freeze-substitution etc.) can be very useful under certain conditions. Fixation
procedures based on water-soluble carbodiimides, periodate-lysine-paraformaldehyde
mixtures and diethylpyrocarbonate vapors have also been suggested. Such fixations, however,
have not yet found widespread application.
We have to keep in mind: in performing their role in tissue stabilization, fixatives will change
the chemical and physical nature of cells, and this leads to denaturation of macromolecules by
additive condensation, intra- and intermolecular cross-linkages or coagulation. Sol-gel
transformations, protein networks, conformational changes are some of the results which can
make the tissue components inaccessible for molecular probes in histochemical studies.

Denaturation by fixation
There is a great variability in protein masking or denaturation among the different tissues by
use of the various fixatives. At least various effects of cross-linkage are obtained with
proteins possessing varying amounts of reactive lysine groups. It can be expected that
different polypeptide chains are linked randomly to give a blend of soluble and insoluble
polymers or copolymers. The final outcome in cells will be further modulated by factors such
as pH, ionic strength and the ratio of fixative to protein.
Differences in reactivity are intrinsic to the different antigenic epitopes under study. It must
be kept in mind that fixation is usually accompanied by alterations of the specific biological
nature, and that the extent of denaturation is always difficult to predict. Whereas the
antigenicity of small peptides like hormones appears to withstand fixation quite well,
proteinous antigens behave capriciously due to unpredictible influence on foldings of the
polypeptide chains and its antigenic structure. Upon the action of aldehydes on antigenic
sites, one can expect changes of immunochemical reactivity due to modification of amino
groups in some antigenic sites and due to conformational changes outside those sites. All
changes may occur at random, thus, fixation is a process which is difficult to control.
Changes of cellular characteristics are regular features in tissue fixation and a compromise
between structural conservation and retention of biological activity must be made. On the
other hand, attempts to reconstitute antigenicity can be tried. For instance, some epitope
retrieval may be achieved by tissue treatments with proteolytic enzymes (f.e. pronase), heat
(microwave), certain buffer solutions or by other formulas (which are usually individual
developments for the tissue under study) which enhance the sensitivity of histological antigen
staining and other molecular probe reactions.

Fixation solutions
Fixatives may be organic or inorganic in nature and irrespective of the classification as
additive or coagulant fixative (see above), the routine laboratory designates fixatives as

Aldehydes.

Organic solvents.

Mercurials.

Oxidizing agents.

Picrates.

Aldehydes fix mainly by cross-linkages and are most widely used for fixation and also well
known from the tanning industry (GUSTAVSON KH, 1956). Then, mixtures of aldehydes (e.g.
paraformaldehyde) with periodate and lysine have been proposed (MCLEAN IW and NAKANE
PK, 1974; HIXSON DC et al., 1981). Organic solvents such as alcohols are protein denaturants
which are mainly employed for cytologic smears but rarely in classical histopathology.
Mercurials fix by a virtually unknown mechanism. Their best application is for fixation of
hematopoietic and reticuloendothelial tissues. Also, the way of fixation of oxidizing reagents
(e.g. permanganate, dichromate and osmium tetroxide fixatives) is not well known. They may
cross-link proteins and cause denaturation of proteins much more extensively than aldehyde
fixatives. Chromic salts form complexes with water and have a cross-linking effects which
are similar to that of aldehydes. Picrate fixatives are based on picric acid with an additive
coagulant mechanism of action. For detailed descriptions of fixative formulations and for
relevant references see also the chapter Fixatives.

Formaldehyde
Since the introduction of formaldehyde by F BLUM and O LOEW as potential fixative of
biological specimens (LOEW O, 1886; BLUM F, 1893; BLUM F, 1894; BLUM F, 1896), this
chemical has become one of the most commonly used fixative for routine histology and
immunohistology. Formaldehyde can be used either alone or in combination with other
chemicals. Though rapidly penetrating into tissue blocks, formaldehyde is not ideally crosslinking as compared with glutaraldehyde.The latter, however, penetrates tissues more slowly
and interferes with macromolecules more intensely than formaldehyde. To achieve
advantages and minimize the disadvantages of both fixatives for an optimal outcome, one can
prepare combinations and vary their concentrations in buffered solutions by trial and success.
Methanol free formaldehyde (preferentially prepared freshly from paraformaldehyde and used
either alone or in combination with other chemical substances) is a widely employed fixative
for immunohistological work. In principle, formaldehyde (HCHO) is a gas and this term
should be in fact restricted to that gas itself. Solutions of formaldehyde gas dissolved in water
are usually called formalin. Yet, the correct use of this terminology is not strictly applied (see
chapter Fixatives).
The molecular mechanisms of tissue fixation with formaldehyde are not well understood. A
number of studies indicate that formaldehyde reacts readily with biological macromolecules
such as proteins, nucleic acids and polysaccharides by the formation of cross-links, methylene
bridges according to BLUM (BLUM F, 1896). The most reactive sites are primary amino groups
(f.e. lysine) and thiols (cystein). Denaturation, intra- and intermolecular cross-links and
polymerization will considerably alter the characteristics of organic structures (FRAENKELCONRAT H et al., 1945; FRAENKEL-CONRAT H et al., 1947; FRAENKEL-CONRAT H and OLCOTT
HS, 1948a, 1948b; FRAENKEL-CONRAT H and MECHAM DK, 1949; HOPWOOD D, 1967;
HOPWOOD D, 1969a, 1969b; 1969d; 1969e; HOPWOOD D, 1970; MAYS ET et al., 1984;
MASON JT and OLEARY TJ, 1991; HELANDER KG, 1994).
Aqueous formaldehyde solutions preserve cellular structures by its reaction with peptides and
nucleic acids; in the presence of calcium, formalin is also useful as fixative for lipids.

Basically, fixation with formaldehyde molecules leads to addition products between aldehyde
and reactive amino groups (primarily with the residues of the basic amino acid lysine) by the
formation of reactive hydroxymethyl groups. Subsequent condensation occurs with other
neighboring amino groups to form methylene bridges between polypeptide chains (methylene
bridge cross-links); some of these reactions are partially reversible. Formalin fixation is
temperature dependent and progressive with time with profound change of macromolecular
conformation by changes of tertiary and quaternary structures of proteins. The final result can
lead to failure of subsequent antibody reactions and other molecular probe reactions.
In the selection of formalin as fixative and in consideration of special staining methods, one
has to reconcile that most of the formaldehyde induced cross-links occur at neutral pH. With
buffered formalin fixatives at neutrality, more cross-links are generated because hydrogen
ions from charged amino groups of protein side chains are dissociated resulting in uncharged
amino groups which contain reactive hydrogen to form the above described addition products.
In contrast, less cross-links occur with acid formalin solutions.

Glutaraldehyde
Fixation of tissue is more efficient with glutaraldehyde than with formaldehyde and relies on
its cross-linking properties. For reproducible results, highly purified glutaraldehyde is needed.
Besides the monomer, glutaraldehyde freqently may contain large amounts polymers, , unsaturated aldehydes after aldol condensation, glutaric acid and inorganic substances (see
BEILSTEINs Handbuch der Organischen Chemie E III 1.3111) which all together can initiate
unexpected reactions. In theses cases and when highly purified preparations are not available,
the degree of purification of glutaraldehyde can be improved by treatment with activated
charcoal and chromatography (e.g. Sephadex G-10), or better, by vacuum distillation over a
Vigreux column; the degree of purity P.I <0.2 (P.I = E235 nm : E280 nm) being strived for.
Methods of purification and standardization for fixation purposes have been published
(FAHIMI HD and DROCHMANS P, 1965; ANDERSON PJ, 1967).
The stabilizing effect is attributed to rapid and persistent intra- und intermolecular crosslinkages of tissue components. Glutaraldehyde is believed to react by a similar mechanism to
formaldehyde. Glutaraldehyde, however, will give a more tightly linked product: its greater
length as compared with formaldehyde and its two aldehyde groups allow glutaraldehyde to
link more distant pairs of protein molecules as formaldehyde. This type reaction is a factor
which makes glutaraldehyde an efficient fixative. The degree of cross-linking is progressive
with time, and depends on the accessibility of -amino groups by aldehyde groups which
leads to the formation of Schiff bases. Important aspects of the reaction of glutaraldehyde on
proteins, the cross-linking and the fixation process have been published (QUIOCHO FA and
RICHARDS FM, 1964; HOPWOOD D, 1967; HABEEB AJ and HIRAMOTO R, 1968; RICHARDS FM
and KNOWLES JR, 1968; HOPWOOD D, 1969a, 1969b, 1969d, 1969e; HOPWOOD D, 1970;
HOPWOOD D et al., 1970; KORN AH et al., 1972; PAYNE JW, 1973; MONSAN P et al., 1975).
Cross-linking fixatives tend to preserve the secondary structure of proteins and may also
protect tertiary structures as well. Yet, it must be kept in mind that this type of fixation can be
accompanied by an alteration of specific biological activity. The extent, however, is difficult
to predict and varies from tissue to tissue and from cell to cell. In the case of antigenic
epitopes, the action of aldehydes will modify one or several amino groups and may change
the reactive regions of the molecule. Thereby, the charge pattern of proteins can be changed
inducing conformational changes. Furthermore, conformational changes outside the antigenic

epitopes must be considered which will also influence protein denaturation. For more details
see chapter Artefactual staining in immunohistology.

Miscellaneous aldehydes
A number of other aldehydes are known and may be used for fixation procedures. For
example acrolein (acrylic aldehyde) which is mainly used in tanning industries. Solutions of
4% acrolein may be employed for histochemistry. This aldehyde produces more cross-links
than formaldehyde, it is, however, unstable at alkaline pH and tends to polymerize into
disacryl when exposed to light.
Glyoxal, malondialdeyhde, 2,3-butanedione and other di- or polyaldehydes are possible
alternatives to the above mentioned aldehydes for the purpose of histological fixation. They
are, however, rarely employed.

Other cross-reacting fixatives

There exist a number of other reagents as protein cross-linking fixatives. They are, however,
not widely employed. From the bulk of possible reagents, just a few are mentioned, e.g.

Water-soluble carbodiimides: compounds that react with and cross-link carboxyl groups.
They form cross-links between soluble proteins by joining C-termini and side chains of
glutamic and aspartic acid units. Carbodiimides are mainly used to prepare conjugates of
peptides and larger proteins.

Diisocyanates and diazonium compounds: reagents often used to conjugate antibodies

with fluorescent labels; in histology used to reduce diffusion artefacts of soluble enzymes
in tissue preparations.

Diimidoesters: theses reagents react rapidly with protein molecules by the formation of
cross-links (amidines). They have proved useful as fixatives in light and electron
microscopy.

Diethylpyrocarbonate: compound consisting of diethyl oxyformate and ethoxyformic

anhydrate and mainly used for cold sterilization of beverages; introduced in histology as a
vapour phase fixative (freeze-dried tissue) for subsequent immunostaining. With
improved water-solubility, this compound can be used as a liquid phase fixative.

Maleimides: reagents with mild cross-linking properties, useful for the preparation of
protein conjugates such as antibodies labeled with enzymes as markers.

Benzoquinones: cross-linking reagents often used in immunochemistry for the

conjugation of enzymes and various protein molecules. Comparable to diimidoesters,
benzoquinones can be applied as fixatives in histology.
Over the years, these reagents have never caught great importance. If the antigenicity of
proteins is critically dependent on free amino groups of an epitope, then carbodiimides can be
an alternative to aldehydes. In the case of light microscopic immunohistology, we prefer
chemically unreactive fixatives like ethanol-acetic acid mixtures (see chapter Fixatives).

Non-aldehyde fixatives
In order to avoid cross-linkage and other aldehyde-induced loss of reactivity of cellular
molecules, other fixatives were looked for as substitutes for aldehydes. Many of them are
coagulating fixatives which precipitate proteins, and ethanol is a typical representative. By
water removal with ethanol, hydrophobic interactions, which give many proteins their tertiary
structure, are (often) disrupted. These events will reduce the solubility of protein molecules
and result in denaturation.
The most common precipitating fixatives are ethanol, methanol and acetone. Acetic acid is
also a denaturant that is used in combination with other precipitating fixatives. Alcohols are
known to cause shrinkage of tissue while acetic acid alone leads to tissue swelling. The
combination of both reagents results in better preservation of morphology. In a number of
histological and immunohistological studies with a variety of organs, the great value of
ethanol-acetic acid fixatives could be clearly shown. Apart from immunohistology, this type
of fixation competed well with conventional formalin fixation with respect to routine
stainings and histochemical reactions (KUHLMANN WD, 1975; WURSTER K et al.,1978;
KUHLMANN WD and PESCHKE P, 2006).
Oxidising fixatives react with various side chains of biomolecules (including proteins) leading
to the formation of cross-links which stabilize the tissue structure. Potassium permanganate,
potassium dichromate, chromic acid and osmium tetroxide are useful in certain specific
histological studies (BAKER JR, 1965a, 1965b; LUFT JH, 1956). Then, osmium tetroxide has
been often used as fixing reagent in light and electron microscopy (SCHULTZE M and
RUDNEFF M, 1865; FLEMMING W, 1895; PORTER KR, 1950; PALADE GE, 1952; PORTER KR
and KALLMAN F, 1953; RYTER A and KELLENBERGER E, 1958; BENNETT HS and LUFT JH,
1959; MILLONIG G, 1961). The effect of some of the above fixatives on proteins has been
described in model experiments (LUFT JH and WOOD RL, 1963; HOPWOOD D, 1969c).
The value of osmium tetroxide for the preservation of cell structure has been already reported
in 1927 (STRANGEWAYS TSP and CANTI RG, 1927). With the development of electron
microscopy in the early 1950s, it became apparent that osmium tetroxide was by far the best
choice for the study of fine structure as compared with the then employed fixatives for light
microscopy (PALADE GE, 1952). The main disadvatage of osmium tetroxide is its slow
penetrability into tissue blocks. Furthermore, the ionic constitution of the buffer influences
the cellular fine structure (TRUMP BF and ERICSSON JLE, 1965) as well as the rate of osmium
penetration. Today, osmium tetroxide has still its important role as secondary fixative in
electron microscopy.
Other fixatives include picric acid and mercuric chloride. Both types of fixatives have proved
useful for special histological studies. The type of fixation, however, is poorly understood.
Recently, new fixative formulations have been proposed as alternatives to aldehydes, organic
solvents etc. with respect to histopathological diagnosis and immunohistology. One of these
promising fixatives is HOPE (HEPES-glutamic acid buffer-mediated organic solvent
protection effect) fixation. This fixative can be employed for cytospins and tissue blocks.
Fixation starts by overnight immersion of fresh tissue specimens into the aqueous protection
solution at low temperature followed by acetone dehydration, paraffin embedment and
microtomy (OLERT J et al., 2001; UMLAND O et al., 2003). Then, special kinds of ionic liquids
containing a heterocyclic cation and based on a substituted imidazole such as 1-methyl-3octyloxymethylimidazolium tetrafluoroborate were shown to be good fixatives with similar
effects as formalin (PERNAK A et al., 2005). All these new fixative formulations are of some

interest as formalin substitutes if they really function as fixatives rather than as tissue
preservatives that prevent autolysis. Further studies will show if those formalin subsitutes
have the advantage of little or no need for antigen retrieval since in many instances the use of
appropriate epitope retrieval allows immunostaining on formaldehyde fixed specimens.

Microwave irradiation for tissue fixation

Fast tissue fixation methods are of interest in the study of precise localization of highly
dynamic cell processes. Fast tissue fixation techniques are also of advantage in diagnostic
histopathology at least for reasons of rapidness. Quick-freezing and freeze-drying
preparations are often successful solutions, but the versatility of these approaches is limited
with respect to simultaneous cytochemical, immunological and conventional stainings in
histology. Hence, the development of other methods are looked for. One promising alternative
with special regard to primary chemical fixatives is fixation of tissues by microwave energy.
Microwaves for tissue fixation were introduced by CP MAYERS (1970). Later on, the basics of
microwave heating patterns in tissue models were described (HAND JW, 1977). The first
approaches focussed on its use for light microscopy, but soon this techniques was also
successfully applied for electron microscopic studies (BERNARD GR, 1974; GORDON HW and
DANIEL EJ, 1974; HOPWOOD D et al., 1984; LOGIN GR and DVORAK AM, 1985; LOGIN GR et
al., 1986; OHTANI H, 1991; LEONG AS, 1993; GIBERSON RT and DEMAREE RS, 1995;
GIBERSON RT et al., 1997). In the meantime, numerous reports on microwave fixation support
the usefulness of this type of fixation for a variety of tissues and purposes (LEONG AS et al.,
1985; LOGIN GR et al., 1991; LOGIN GR et al., 1992; LOGIN GR and DVORAK AM, 1994a,
1994b; TINLING SP et al., 2004).
Microwaves are a form of non-ionizing radiation which may be generated by domestic
microwave ovens at a frequency of 2.5 GHz and 600 Watt output. The exposure of dipole
molecules such as water or the polar side chains of proteins to the electromagnetic field
results in oscillation corresponding to the frequency of the generator. Immediately, the
kinetics result in heat which is proportional to the energy flux until radiation ceases; the heat
being controlled by the energy level and the duration of exposure. The optimal temperature
for fixation is in the range of 60C and 85C. Temperaure and time are usually establised for
each tissue.
The heat in microwave fixation is probably the primary factor in tissue fixation. Yet, the rapid
movement of molecules (induced by the oscillating electromagnetic field) will also have a
direct influence on the outcome of fixation. In the case of combined fixation by microwaves
and aldehydes as cross-linking reagents, the diffusion rate of the chemical fixative is
enhanced and will accelerate the process of cross-linkage (LOGIN GR et al., 1986).
Furthermore, other still unknown mechanisms are expected to occur with irradiation.
Influences on non-covalent secondary bonding, hydrogen bonds, van der Waals interactions
etc or disruption of bound water are possible which all together can alter biological systems.

Selected publications for further readings

Schultze M and Rudneff M (1865)
Loew O (1886)

Blum F (1893)
Blum F (1894)
Flemming W (1895)
Blum F (1896)
Strangeways TSP and Canti RG (1927)
Fraenkel-Conrath H et al. (1945)
Fraenkel-Conrath H et al. (1947)
Fraenkel-Conrath H and Olcott HS (1948a, 1948b)
Fraenkel-Conrath H and Mecham DK (1949)
Porter KR (1950)
Palade GE (1952)
Porter KR and Kallman F (1953)
Gustavson KH (1956)
Luft JH (1956)
Ryter A and Kellenberger E (1958)
Bennett HS and Luft JH (1959)
Millonig G (1961)
Luft JH and Wood RL (1963)
Malamed S (1963)
Robertson JD et al. (1963)
Sabatini DD et al. (1963)
Quiocho FA and Richards FM (1964)
Sabatini DD et al. (1964)
Anderson DR (1965)
Baker JR (1965a, 1965b)
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Karnovsky MJ (1965)
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Maunsbach AB (1966a, 1966b)
Anderson PJ (1967)
Charret R and Faur-Fremiet (1967)
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Mayers CP (1970)
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Korn AH et al. (1972)
Kuhlmann WD and Viron A (1972)
Payne JW (1973)
Bernard GR (1974)
Gordon HW and Daniel EJ (1974)
McLean IW and Nakane PK (1974)
Glauert AM (1975)

Kuhlmann WD (1975)
Monsan P et al. (1975)
Hand JW (1977)
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Ohtani H (1991)
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Full version of citations in chapter References.

Prof. Dr. Wolf D. Kuhlmann, Heidelberg

20.08.2009