Fixative Penetration Rate

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Fixative Penetration Rate

Appendix A: Penetration and Fixation Rates of formaldehyde

Formaldehyde is one of the most rapidly penetrating fixatives used. Unfortunately, it is one of the
slowest to fix the tissue. This paradox was finally explained by
Burnett
1
in 1982.

An excellent description of the properties of formaldehyde may be found in John Kiernan’s book.
2

The penetration rate of formaldehyde in mm/hr is a variable thing. It depends on how the data is
obtained. It may also vary slightly depend
ing on tissue type. The penetration rate of formaldehyde
fixatives has been extensively studied, often with conflicting results. The penetration of non
-
coagulating fixatives is difficult to measure.

The original experiments of Medawar
3
utilized plasma clo
ts with an indicator to mark depth of
penetration. Medawar showed that fixatives obey the diffusion laws, that is, the depth penetrated was
proportional to the square root of time. Medawar determined a coefficient of diffusibility for each
fixative, the Me
dawar constant
K
.

Using the equation
d = K√t
, where
d
is distance penetrated in mm,
t
is time in hours and
K
the
Medawar constant for the fixative in question, it is possible to determine the penetration rate.

Medawar determined
K
= 5.5 for formaldehyde.

Using this value NBF would penetrate 27.5 mm in 25
hours. Plasma clots are easier to penetrate than solid tissues, so the rate is probably less.

Baker
4
chose a gelatin/albumen gel to more closely mimic solid tissue and determined

K
= 3.6 for formaldehyd
e, or 18 mm in 25 hours. Baker also pointed out that the actual penetration
into tissue would probably be less, possibly due to the resistance of lipid containing cell membranes.
He quotes the data of Tellyesnicsky
5
who, mainly using liver tissue samples,
indicated a more
conservative
K
= 0.78 for formaldehyde. That would translate to 3.9 mm in 25 hours.

From
d = K√t
, it follows that fixatives penetrate more quickly into small samples of tissue compared
to large ones. The initial rate of penetration into t
issue is extremely rapid.

The first layer of cells (20 μm) takes a second or so (70 mm/hr). Using Baker’s
K
=3.6, the following
examples will illustrate further;

1 hour = 3.6 mm,

4 hours = 7.2 mm, averaged to (1.8mm/hr),

9 hours = 10.8 mm (1.2mm/hr),

16 hours = 14.4 mm (0.9mm/hr),

25 hours = 18 mm (0.72mm/hr),

100 hours = 36 mm (0.36mm/hr).

So much for the penetration rate, the real issue is the fixation rate, i.e. penetration rate plus binding
time.

Fox et al.
6
used
14
C labeled formaldehyde to stu
dy the covalent binding time for rat kidney tissues. At
a temperature of 25°C, the amount of formaldehyde bound to tissue increased with time until
equilibrium was achieved at 24 hours. At 37°C the reaction was faster and equilibrium was reached at
18 hour
s. A later study by Helander
7
also used
14
C labeled formaldehyde to study binding time for the
fixation of rabbit liver. At 25°C. equilibrium was achieved at 25 hours.

7 Immunopathology Laboratory Eastern Health Hospitals On
-
site Consultation


2007
-
12
-
07


The correlation of results between these two studies is impressive. Particularly in view of the fact that
Fox used
16
μ
m
thick sections of fresh rat kidney, whereas Helander used
4 mm
cubes of fresh rabbit
liver. The virtually identical equilibrium times
achieved by each study indicate that penetration time is
not a factor in the kinetics of the reaction. Despite the fact that thin slices of tissue will be penetrated
faster than thicker cubes, it would seem that the binding time is the limiting factor for
tissue
stabilization. In a further study also by Helander,
8
using rat brain and kidney, equilibrium was not
achieved until 50 hours. However, the tissue in Helander’s

latest study was twice the thickness (8mm)
of the original study, a factor to be taken into account when comparing the data.

Failure to recognize the importance of formaldehyde binding time is the leading cause of the
tremendous intra and inter
-

laborato
ry variability in immunohistochemical (IHC) performance. A
clinical laboratory’s so called ‘routine formaldehyde fixation’, actually consists of allowing the tissue
to fix for variable periods of time, dictated by the start time of the tissue processor!

F
ormaldehyde fixes not by coagulation, but by addition, reacting with basic amino acids (primarily
lysine and arginine) to form several adducts. These reactions are readily reversible by water and
alcohol. These adducts have free hydroxymethyl groups which
are capable of further reaction to form
stable methylene bridges between proteins (see Kiernan
2
for more information). This type of cross
-
linking is responsible for the stabilization of proteins that we term fixation.

For both 1 mm thick core biopsies and

4mm thick tissue slices, the minimum stabilization time is 24
-
25 hours at ambient temperatures. The minimum stabilization time does not, unfortunately, denote
complete fixation time. The initial cross
-
links are still relatively weak and easily reversible;

stronger
cross
-
linking continues to occur over time. Complete fixation is thought to take at least 7 days. Even
after this time cross
-
links continue to form slowly.

Werner,
9
quoting the two papers above, considers cross
-
linking complete in 24

48 hours, b
ut also
expresses concern about the ‘over
-
fixation’ due to excessive cross
-
linking, which may occur if
fixation is allowed to exceed 24

48 hours. I agree with Werner, in that cross
-
linking may mask some
epitopes, but in my experience this does not occur wi
th the vast majority of antibodies in use until 5

7
days of fixation. Even then, providing the IHC has been optimized, with the majority of antibodies,
fixation up to 4 weeks is acceptable. The major strength of formaldehyde, as a fixative for IHC, lies in

the fact that the cross
-
linking is 90% reversible. This reversibility allows the successful use of
‘Antigen retrieval’ techniques. Far more serious is the problem of short <24 hour fixation.

In aqueous solution, formaldehyde rapidly becomes hydrated to f
orm methylene hydrate (methylene
glycol).
1
The equilibrium of the reaction lies so far in favor of the hydrated form, that little(less than
0.1%) true formaldehyde is present.
6
The reactivity of aqueous solutions of formaldehyde is known to
physicals chemi
sts as an example of a “clock” reaction. The conversion of methylene glycol to
formaldehyde by removal of the little formaldehyde present can be used as a “real
-
time” clock,
measured in hours.
1

Formaldehyde fixation begins at the periphery of the tissue.
The initial layers of cells bind all of the
available formaldehyde (<0.1%) and start the ‘clock’. Methylene glycol continues to rapidly penetrate
the tissue and, over hours, more formaldehyde is generated from methylene glycol. If this process is
interrupt
ed before completion, the formation of addition compounds will be incomplete, easily
reversed and full stabilization by cross
-
linking will not occur. Depending upon the time of
interruption, the periphery may show adequate cross
-
linking, whereas the remain
der of the tissue is
fixed by coagulant alcohol during processing. This may have disastrous effects upon IHC staining.
This will occur whether the tissue is a small biopsy or a 4 mm slice.

8
Immunopathology Laboratory Eastern Health Hospitals On
-
site Cons
ultation


2007
-
12
-
07 9

References


1. Burnett MG. The mechanism of the formaldehyde clock reaction: methylene glycol dehydration. J
Chem. educ. 1982;59:160.



2. Kiernan JA. Histological and Histochemical Methods: Theory and Practice, 3
rd
Edition, 1999.
Oxford: Butterworth
-
Heinemann. ISBN # 0
-
7506
-
3106
-
6.



3. Medawar PB. The rate of penetration of fixatives. JR Microsc Soc. 1941;61:46



4. Baker JR. Principles of Biological Microtechnique, Methuen & Co. Ltd. 1958. pp 37

40.



5. Tellyes
nicsky K. Article on ‘Fixation’ in R. Krause’s Enzyklopädie der mikroskopischen Technik,
vol.2. Berlin (Urban & Schwarzenberg). 1926.



6. Fox CH, et.al. Formaldehyde fixation. J Histochem. Cytochem. 1985;33:845

53



7. Helander KG. Kinetic studies of fo
rmaldehyde binding in tissue. Biotechnique and Histochemistry.
1994; 69:177

179.



8. Helander K.G. Formaldehyde binding in brain and kidney: a kinetic study of fixation. The Journal
of Histotechnology. 1999;22(4):317

8.



9. Werner M., et.al. Effect of
formalin fixation and processing on immunohistochemistry. Am J Surg
Pathol. 2000;24(7):1016

9.


Fixation and fixatives

Anthony S
-
Y Leong

Introduction

Once

tissues are removed from the body, they undergo a process of self
-
destruction or
autolysis which is initiated soon after cell death by the action of intracellular enzymes
causing the breakdown of protein and eventual liquefaction of the cell. Autolysis is

independent of any bacterial action, retarded by cold, greatly accelerated at temperatures of
about 30

C and almost inhibited by heating to 50

C.

Autolysis is more severe in tissues which are rich in enzymes, such as the liver, brain and
kidney, and is le
ss rapid in tissues such as elastic fibre and collagen. By light microscopy,
autolysed tissue presents a `washed
-
out' appearance with swelling of cytoplasm, eventually
converting to a granular, homogeneous mass which fails to take up stains. The nuclei of
autolytic cells may show some of the changes of necrosis including condensation (pyknosis),
fragmentation (karyorrhexis) and lysis (karyolysis) but these are not accompanied by an
inflammatory or cellular response. There may be diffusion of intracellular s
ubstances of
diagnostic significance, such as glycogen which is lost from the cells in the absence of
prompt and suitable fixation. Autolysis also causes desquamation of epithelium which
separates from its basement membranes.

Bacterial decomposition can al
so produce changes in tissues that mimic those of autolysis
and is brought about by bacterial proliferation in the dead tissue. Such bacteria may normally
be present in the body during life such as the non
-
pathogenetic organisms present in the
bowel, or ma
y be present in diseased tissues at the time of death such as in septicaemia.

The objective of fixation is to preserve cells and tissue constituents in as close a life
-
like state
as possible and to allow them to undergo further preparative procedures witho
ut change.
Fixation arrests autolysis and bacterial decomposition and stabilises the cellular and tissue
constituents so that they withstand the subsequent stages of tissue processing. Aside from
these requirements for the production of tissue sections, in
creasing interest in cell
constituents and the extensive use of immunohistochemistry to augment histological
diagnosis has imposed additional requirements. Fixation should also provide for the
preservation of tissue substances and proteins. Fixation is, th
erefore, the first step and the
foundation in a sequence of events that culminates in the final examination of a tissue section.

It is relevant to point out that fixation in itself constitutes a major artefact. The living cell is
fluid or in a semi
-
fluid s
tate, whereas fixation produces coagulation of tissue proteins and
constituents, a necessary event to prevent their loss or diffusion during tissue processing; the
passage through hypertonic and hypotonic solutions during tissue processing would otherwise
disrupt the cells. For example, if fresh unfixed tissues were washed for prolonged periods in
running water, severe and irreparable damage and cell lysis would result. In contrast, if the
tissues were first fixed in formalin, subsequent immersion in water
is generally harmless.

A large variety of fixatives is now available but no single substance or known combination of
substances has the ability to preserve and allow the demonstration of every tissue component.
It is for this reason that some fixatives hav
e only special and limited applications, and in
other instances, a mixture of two or more reagents is necessary to employ the special
properties of each. The selection of an appropriate fixative is based on considerations such as
the structures and entitie
s to be demonstrated and the effects of short
-
term and long
-
term
storage. Each fixative has advantages and disadvantages, some are restrictive while others are
multipurpose. The requirements of a large through
-
put diagnostic laboratory are also quite
diffe
rent from those of a research laboratory with small numbers of specimens for specialised
structural analysis and less requirement for urgency.

Over the years, various classifications of fixatives have been proposed, with major divisions
according to functi
on as coagulants and non
-
coagulants, or according to their chemical nature
into three general categories which include alcoholic, aldehydic and heavy metal fixatives. A
modification of Hopwood's classification
1

shown below will be adopted in this chapter.



Aldehydes, such as formaldehyde, glutaraldehyde.



Oxidising agents: metallic ions and complexes, such as osmium tetroxide, chromic
acid.



Protein
-
denaturing agents, such as acetic acid, methyl alcohol (methanol), ethyl
alcohol (ethanol).



Unknown mechanism, s
uch as mercuric chloride, picric acid.



Combined reagents.



Microwaves.



Miscellaneous: excluded volume fixation, vapour fixation.

Fixatives

Aldehydes and other cross
-
linking fixatives

FORMALDEHYDE

Formaldehyde, as 4% buffered formaldehyde (10% buffered forma
lin), is the most widely
employed universal fixative particularly for routine paraffin embedded sections. It is a gas
with a very pungent odour, soluble in water to a maximum extent of 40% by weight and is
sold as such under the name of formaldehyde (40%)
or formalin (a colourless liquid).
Formaldehyde is also obtainable in a stable solid form composed of high molecular weight
polymers known as paraformaldehyde. Heated paraformaldehyde generates pure gaseous
formaldehyde which, when dissolved in water, reve
rts mostly to the monomeric form.
Aqueous formaldehyde exists principally in the form of its monohydrate, methylene glycol,
CH
2
(OH)
2
, and as low molecular weight polymeric hydrates or polyoxymethylene glycols. It
has been suggested that the hydrated form,
methylene glycol, is the reactive component of
formaldehyde but this has been disputed
2

(
Fig. 1
).

Four per cent formaldehyde or 10% buffered formalin is commonly prepared by adding 100
ml of 40% fo
rmaldehyde to 900 ml distilled water with 4 g sodium phosphatase, monobasic
and 6.5 g sodium phosphate, dibasic (anhydrous). To be effective, the specimen should be
completely submerged in five to ten times its volume of fixative.

Ten per cent buffered neu
tral formalin preserves a wide range of tissues and has the
advantage of being a forgiving fixative. It requires a relatively short fixation time but can also
be used for long
-
term storage as it produces no deleterious effects on tissue morphology with
nuc
lear and cytoplasmic detail being adequately preserved.

Details of the fixing action of formaldehyde and other aldehydes are not known although the
general principles are understood. It is thought that the aldehydes form cross
-
links between
proteins, creat
ing a gel, thus retaining cellular constituents in their in vivo relationships to
each other. Soluble proteins are fixed to structural proteins and rendered insoluble, giving
some mechanical strength to the entire structure which enables it to withstand su
bsequent
processing. With the aldehydes, cross
-
links are formed between protein molecules, the
reaction being mostly with basic amino acid lysine, although other groups such as imino,
amido, peptide, guanidyl, hydroxyl, carboxyl, SH and aromatic rings may
also be involved.
2

Only those lysine residues which are on the exterior of the protein molecule react, these
usually accounting for 40
-
60% of the total lysyl residues.

Although the extent of denaturation produced by fixation this does not matter greatly i
n
routine tissue pathology, it is of particular importance in the detection of antigens both by
immunofluorescence and immunoenzyme techniques as well as in high resolution electron
microscopy. Similarly, the shapes of large molecules must not be changed i
f they are to be
recognised by biochemical analysis. Glutaraldehyde causes a loss of up to 30% of the alpha
helix structure of protein, depending on the type of protein. Fixation with osmium tetroxide
or post osmication of glutaraldehyde
-
fixed material cau
ses the complete denaturation of
protein.

Formalin does not precipitate proteins and only slightly precipitates other components of the
cell. It does not harden or render albumin insoluble but subsequent hardening by alcohols is
prevented. Formalin neither

preserves nor destroys adipose tissue and is a good fixative for
complex lipids but has no effect on neutral fats. Although not the fixative of choice for
carbohydrates it preserves proteins so that they hold glycogen which is otherwise readily
leeched fr
om the cell.

Formaldehyde solution is nearly always acid. It certainly becomes acid on storage as formalin
oxidises to formic acid, reducing its preserving capabilities such that neutralisation of the
solution is a requirement. In addition, formaldehyde so
lution produces acid formalin
haematin pigment which can be seen in sites containing blood. If calcium carbonate is used
for neutralising formalin the resultant solution does not retain its neutral pH and calcium
carbonate itself can deposit in tissues, le
aving areas of `pseudocalcification'. Phosphate
buffers such as sodium phosphate monobasic and sodium phosphate dibasic are effective for
the neutralisation of formalin and the pH of the solution produced is stable for many months.
Formalin should not be u
sed with chromates because it readily oxidises to formic acid.

A concentrated solution of formalin sometimes becomes turbid on storage through the
production of paraformaldehyde which decreases the strength of the solution, but can still be
used as a fixat
ive following filtration. Formalin favours the staining of acidic structures
(nuclei) with basic dyes and diminishes the effect of acid dyes on basic structures
(cytoplasm).

Formaldehyde is an immediate irritant to the eyes, upper respiratory tract and the

skin, and
safety precautions should include proper ventilation and exhaust, limited or restricted
exposure periods and thorough washing if spilt on tissue surfaces such as the skin.

GLUTARALDEHYDE

Like formaldehyde which acts through the formation of cros
s
-
links between protein end
-
groups, glutaraldehyde has also been used extensively as an agent for protein
-
protein linkage
and hence for fixation. An aqueous solution of glutaraldehyde (glutaric dialdehyde) is a
complex mixture at room temperature, consisti
ng of approximately 4% free aldehyde, 16%
monohydrate, 9% dihydrate and 70% hemiacetal. Free glutaraldehyde may form polymers, or
a monohydrate and a dihydrate, which may cyclize to give a hemiacetal which in turn may
also polymerise. Some favour the polym
er as the reactive species while others suggest that
pure, monomeric, glutaraldehyde is the best fixative and much less inhibitory to enzymes
than is the mixed monomeric
-
polymeric product. The success of glutaraldehyde as a cross
-
linking agent may also dep
end on the large range of different molecules present
simultaneously in the fixation solution.

When glutaraldehyde solutions are kept for long periods at ambient temperatures, there is a
tendency for precipitates to form and for aldehyde

levels to fall so that some method of
purification may be required. Glutaraldehyde may be purified to the monomeric form by
removing oligomers, polymers and other impurities through simple shaking with barium
carbonate, vacuum distillation or treatment wi
th activated charcoal
3

and chromatography on
Sephadex G
-
10 has produced equally good results. Vacuum distillation after prior treatment
of commercial glutaraldehyde solutions with sodium chloride and ethanol has become the
most widely used technique for pu
rification.
4

There are many variations in the preparation of this fixative, including the percentage of
glutaraldehyde, additives, and buffers. Because of its low penetration, only small blocks of
tissues (1
-
2 mm
3
) fix well at temperatures of 1
-
4

C. The
fixed tissue specimen can be stored
in buffer solution for many months.

The slow penetration, the requirement for cold temperature and the need for a storage
medium, have greatly limited the use of glutaraldehyde in histology. It is, however, the most
wide
ly used fixative for standard electron microscopy.

Other uses for glutaraldehyde all of which rely its cross
-
linking properties include the
preparation of tissue xenografts, particularly cardiac valves, chemical sterilisation and
disinfection. Glutaraldehy
de has an inhibitory effect on catalase
5

allowing the selective
demonstration of the peroxidase activity of peroxisomes.

MISCELLANEOUS ALDEHYDES

Acrolein (acrylic aldehyde) is mainly used in the tanning industry. It produces more cross
-
links than formaldeh
yde under optimal conditions but is unpleasant to use and unstable at
alkaline pH levels. Acrolein has a tendency to polymerise into disacryl, a solid plastic when
exposed to light. It has been employed as a fixative for enzyme cytochemistry as labile
enzy
mes like glucose
-
6
-
phosphatase are retained in tissue fixed in 4% acrolein.

Glyoxal (ethanedial, diformyl), malonaldehyde (malonic dialdehyde), diacetyl (2,3
-
butanedione) and the polyaldehydes are other aldehydes which have been infrequently
employed for f
ixation, mostly for special situations, to retain specific enzymes or proteins for
histochemistry. In terms of effectiveness as cross
-
linking agents glutaraldehyde is the most
efficient although acrolein, when present in excess, is nearly as efficient and
succinic
dialdehyde is also comparable.

OTHER CROSS
-
RECTING FIXATIVES

Many other reagents are available for use as protein cross
-
linking fixatives although these are
not widely employed in histology.

Chloro
-
s
-
triazides or cyanuric chloride has been used fo
r the preservation of mucins in rat
salivary glands
6

and for immunofluorescence studies.
7

Carbodiimides are compounds which react with a carboxyl and an amino group with
elimination of water to give a peptide and the corresponding urea. This reaction can b
e used
for cross
-
linking soluble proteins, such as the linkage of small peptides to larger proteins like
serum albumin to provide complexes suitable as antigenic stimuli, but they are seldom
employed as fixatives.

Diisocyanates are used for introduction of

fluorescent labels into proteins. Diazonium
compounds as stabilised salts have been used to reduce diffusion of soluble enzymes from
tissue sections and salts such as Fast Garnet GBC, Fast Red B, and Fast Blue R were
effective in fixing an otherwise solub
le aminopeptidase in rat kidney.
8

Diimido esters react rapidly with proteins forming cross
-
links between amino groups to result
in amidines which are stable to acid hydrolysis. These esters have been used as fixatives for
electron microscopy
9
-
10

and immuno
histochemistry.
11

Diethylpyrocarbonate is a compound consisting of diethyl oxydiformate and ethoxyformic
anhydrate which is employed for cold sterilisation particularly of alcoholic beverages. This
compound reacts with tryptophan to quench background fluor
escence induced by aromatic
residues. It was first used as a vapour phase fixative for freeze
-
dried blocks to preserve
antigenic determinant sites for proteins and peptides. It has also been proposed as a liquid
phase fixative for small blocks, in phosphat
e buffer at pH 6.0, especially if its solubility is
improved with the addition of small quantities of ethanol.

Maleimides, a number of N
-
substituted bismaleimides, synthesised as sulphydryl reagents,
possess mild cross
-
linking properties for proteins. Last
ly, benzoquinone, a compound
unsaturated ketone, reacts with amines, amino acids and proteins to give various additional
products. It has been used as a fixative for peptide antigens in various endocrine tissues, in
both vapour and liquid phase.
11

Oxidisin
g agents: metallic ions and complexes

Much less is known of how metallic ions and oxidising agents react with proteins.

OSMIUM TETROXIDE

The most commonly used metallic ion in fixation is osmium tetroxide which was initially a
tissue fixative used in cytol
ogy, but poor penetration limited its application in light
microscopy. It is now largely employed as a secondary fixative in electron microscopy.
Osmium tetroxide is known to form cross
-
links with proteins as reflected in the rapid increase
in viscosity of

a protein solution when they react together, however, there is very little
additional information as to its the mechanism of action. There is some general agreement
that osmium tetroxide reacts with unsaturated lipids as it is reduced with the formation o
f
black compounds containing hexavalent osmium. Various hypotheses of lipid stabilisation
have been postulated and these include the oxidation of double bonds between adjacent
carbon atoms to form monoesters and diesters, the binding of lipid to protein an
d the
conversion of unsaturated fatty acids to stable glycol osmates. More recent studies show that
the reaction of osmium tetroxide is largely with lipid rather than protein.

Osmium tetroxide is used for preservation of fine structures in electron micros
copy and is
effective for small (2
-
3 mm
3
) specimens. While vapours of this fixative will preserve blood
and tissue smears, its low and uneven penetration limits its application in routine light
microscopy and osmium tetroxide fixed tissues often crumble if

embedded in paraffin.
Osmium tetroxide also interferes with many staining procedures.

CHROMIC ACID

Chromic acid (chromium trioxide) is a strong oxidiser that is used with other ingredients. It
has no effect on fats, penetrates slowly and leaves tissues in

a state where shrinkage may
occur during subsequent processing. Chromium salts form complexes with water which
combine with reactive groups of adjacent protein chains to bring about a cross
-
linking effect
similar to that of formalin. The reaction of potas
sium dichromate with adrenal medullary
catecholamines results in the production of black or brown water
-
insoluble precipitates. The
dichromate
-
oxidation product is not only visible grossly but also in the tissue section and is
still regarded as a rapid mea
ns of identifying tissues with aromatic amines such as adrenal
medullary tumours. Potassium dichromate is never used alone and, if employed other than for
the demonstration of amines, should be washed thoroughly to remove the oxide that forms as
it cannot
be removed later in processing.

Other heavy metals such as palladium chloride and uranium may result in some degree of
tissue fixation but have no practical application in histopathology.

Protein
-
denaturing agents

The structure of proteins is largely dependent on the arrangement of covalent bonds in the
sequence of amino acids forming the peptide chain, and hydrogen bonding between the
components of the peptide chain itself and side chains; these forming the primary

and
secondary structures of a protein respectively. The tertiary structure (the total structure in
three dimensions), results from ionic or electrostatic bonds (between the basic and acidic
amino
-
acid residues of peptides), disulphide bonds and hydrophobi
c bonds (between
hydrocarbon
-
like side chains of leucine, isoleucine, valine, phenylalanine, tryptophan and
tyrosine) which are preferentially situated in the relatively water
-
free interior of the protein
molecule. These forces contribute to the exclusion
of water from the peptide backbone and
are relatively protected from reagents dissolved in the medium. Hydrophobic bonds are weak
but as some 30% of the amino acids in a protein will have non
-
polar side chains, the total
effect of hydrophobic bonds is cons
iderable.

METHYL AND ETHYL ALCOHOL

Alteration of the structure of proteins brought about by methanol and ethanol is primarily due
to disruption of the hydrophobic bonds which contribute to the maintenance of the tertiary
structure of proteins. Hydrogen bo
nds appear to be more stable in methanol and ethanol than
in water so that while affecting the tertiary structure of proteins, these alcohols may preserve
their secondary structure.

Methanol and ethanol are the only alcohols which have a role as fixatives.

Methanol is
closely related in structure to water and it competes almost as effectively as the latter for
hydrogen bonds. Ethanol is also closely related in structure and both replace water molecules
in the tissues, unbound as well as bound, during fixati
on.

While absolute ethanol preserves glycogen, it can cause distortion of nuclear detail and
shrinkage of cytoplasm. If fixation is prolonged, the alcohols remove histones from the nuclei
and later extract RNA and DNA.

Methacarn, a 6:3:1 mixture of absolut
e methanol, chloroform and glacial acetic acid has been
used for the preservation of helical proteins in myofibrils and collagen. More recently it has
been used as the fixative of choice for the demonstration of intermediate filaments by
immunohistochemica
l techniques.

ACETIC ACID

Acetic acid is never used alone but is often combined with other fixatives that cause
shrinkage such as ethanol and methanol. Acetic acid penetrates thoroughly and rapidly but
lyses red blood cells.

Unknown mechanisms

PICRIC ACID

Picric acid, when used in combination with other ingredients, leaves tissue soft and penetrates
well, precipitating all proteins. It will continue to react with the tissue structures and cause a
loss of basophilia unless the specimen is thoroughly washed
following fixation.

MERCURIC CHLORIDE

Mercuric chloride (corrosive sublimate, bichloride of mercury) and other salts of mercury
were common histological fixatives in the past. These penetrate rapidly and precipitate all
proteins, reacting with a number of
amino acid residues including thiol, amino, imidazole,
phosphate and hydroxyl groups. The production of hydrogen ions makes the fixative solution
more acidic and mercuric crystals deposited in the tissue need to be removed before staining.
Mercuric chlorid
e is contained in Zenker's, Helly's, Ridley's and B5 solutions. It should also
be noted that mercuric salts are highly toxic and must not be disposed into sewerage systems.
One method of disposal is to precipitate the mercuric chloride with thioacetamide.
For
example, mixing 1 litre of Zenker's solution with 20 ml of 13% thioacetamide solution in a
tightly capped container results in a precipitate of mercuric sulphide which can be filtered out
and disposed of safely.

ACETONE

Acetone is a clear, colourless,
inflammable liquid which is miscible with water, ethanol and
most organic solvents. It has been used as a dehydrating agent in tissue processing and is
more volatile than alcohols and other dehydrants. It has a rapid action but causes brittleness
in tissue

if exposure is prolonged and because it is volatile and inflammable, acetone is not
used in automated processing schedules. However, it has a greater solvent action on lipids
and is rapidly removed by most clearing agents, making it very useful in manual
processing
procedures.

More recently, acetone has been employed as a fixative in the acetone
-
methylbenzoate
-
xylene (AMEX) technique.
12

This requires overnight fixation of tissues in acetone at
-
20

C
then clearing in methylbenzoate and xylene before paraffi
n embedding. The product is
claimed to show better histologic preservation than is possible to obtain in frozen sections,
yet retaining reactivity for labile lymphocyte membrane antigens.

Microwave (MW) irradiation

MW FOR PRIMARY TISSUE FIXATION

Microwave
irradiation for tissue fixation was first introduced by Mayers,
13

who reported that
direct exposure to MWs generated by a 630
-
watt heating device, produced satisfactory
fixation in both mouse and human post mortem tissues. Subsequently, a more extensive st
udy
on the effects of MWs on whole carcases of hairless mice, showed that morphological
preservation of various tissues depended upon generation of an optimal temperature for each
tissue, which ranged between 70

C and 85

C.
14

Heating above or below these
t
emperatures produced various artefacts such as vacuolation and changes in chromatin
pattern. Bernard
14

was probably the first to indicate that MWs had potential applications in
electron microscopy, although he himself was unable to achieve optimal organell
e
preservation. Heat has been used to induce the partial denaturation of protein but never
gained popularity as a method of fixation. This is largely due to the difficulties of controlling
conventional forms of heating such as a naked flame, steam, or a wa
ter bath, all of which do
not result in uniform heating.

MWs are a form of non
-
ionising radiation, commonly generated by domestic ovens at a
frequency of 2.5 GHz. The exposure of dipolar molecules such as water and polar side chains
of proteins to the rapi
dly alternating electromagnetic fields results in oscillation through
180


at the rate of 2.5 billion cycles per second. The molecular kinetics induced result in
instantaneous heat which is proportional to the energy flux and continues until radiation
ceas
es. Heating by MWs thus offers a method of overcoming the limitations imposed by the
normally poor heat conducting properties of biological tissues. Microwaves of 2.5 GHz
penetrate several centimetres into biological material and the heat produced can be c
ontrolled
by adjustment of the energy levels and the duration of exposure. More recently, it has become
recognised that other molecules with an uneven distribution of electrical charge or
asymmetrical molecular configuration, such as inorganic material and

copper oxides, can also
move in the electromagnetic field. As such, MWs have been used to melt inorganic materials
and to generate mixed copper oxides for the production of super conductors.

Although heat is currently considered to be the primary factor r
esponsible for many of the
effects of MWs in tissue fixation, processing, and staining, the rapid movement of molecules
with the electromagnetic flux may itself have a direct role. While heat or thermal energy will
increase molecular kinetics and hasten mo
lecular reactions, the rapid movement of molecules
directly induced by the oscillating electromagnetic field will give rise to increased collision
of molecules and, in turn, accelerate chemical reactions.

In the case of MW accelerated fixation with cross
-
l
inking aldehyde solutions, the fixating
agents are generally present as oligomers and required degradation to monomers or dimers.
The diffusion of these monomers or dimers is enhanced by exposure to MWs. Viscosity, and
hence diffusion rates in liquids can
vary considerably with temperature, and temperature
influences the penetration of tissues by fixing reagents.
15

A

number of other physical
mechanisms have been hypothesised to occur with MW irradiation. Field
-
induced alterations
in macro
-
molecular hydrogen bonding, proton tunnelling and disruption of bound water may
induce alterations in biologic systems. Although th
e proton energy generated by MWs is too
small to alter covalent bonds, low intensity MW fields may readily affect the integrity of non
-
covalent secondary bonding, including hydrophobic interactions, hydrogen bonds, and van
der Waals' interactions that make

up the precise steric interactions at the cell membrane.
16

Besides the fixation of whole mice
14

and rabbit fetuses,
17

Leong and Duncis
18

investigated the
feasibility of using MWs to fix large biopsy specimens and viscera. MWs have limited
penetration in s
olid tissues and temperature gradients of up to 15

C were observed between
the surface and core of large pieces of solid tissue such as the spleen, kidney, breast and liver.
Because it is impossible to raise the tissue core temperature to an optimal level
to accomplish
fixation without over
-
heating the surface, the irradiation has to be done in two phases. Initial
irradiation of large specimens such as the stomach, solid organs, and segments of bowel is
done primarily to render the tissues sufficiently firm

to allow easy handling and dissection.
This can be accomplished by completely immersing the specimen in a volume of normal
saline and irradiating it to a saline temperature of 68
-
74

C. This procedure is sufficient to
harden the specimen and eliminates the

need to pin
-
out the viscera or to slice the tissue for
overnight fixation in formaldehyde as is the convention. Specimens hardened by irradiation
have the added advantage of retaining much of their natural colour and pliability. Dissection
of lymph nodes
in particular, is made easier as they become opaque after irradiation and their
pink
-
tan colour contrasts sharply against bright yellow fat tissue. Because of the limited
penetration of MWs and the temperature gradient between surface and deeper regions of

large specimens, first stage fixation is uneven and not optimal. Therefore, fixation should be
completed after sampling by immersing the 2
-
3 mm thick tissue blocks in saline and
irradiating to a temperature of 50
-
68

C as described below.
18

Domestic MW ove
ns operating at 2.45 GHz and at 600 Watt output produce satisfactory
fixation of most tissues by irradiation in normal saline to a temperature of 50
-
68

C. When
only a small volume of tissue is irradiated, the procedure can be accomplished in about
120

seco
nds. MWs can thus be used in diagnostic laboratories as the primary method of
routine fixation.
19
-
20

Although any domestic oven may be used, those with a rotating plate or
carousel give more even distribution of the electromagnetic waves and a digital time
r allows
greater accuracy. Laboratory
-
grade ovens are commercially available, but are considerably
more expensive and offer no advantage over domestic ovens which are easy to calibrate since
the temperature attained is proportional to the duration of elect
romagnetic flux.
20

The
availability of temperature probes in some domestic ovens further simplifies operation as
these allow a choice of temperature settings.

MW fixation does not have any deleterious effect on special stains. It has also been shown
that t
issue antigens are often better preserved in MW irradiated tissue than those fixed
routinely in 10% formalin and processed in the usual manner.
21

The speed with which MWs can accomplish fixation of both large and small biopsy
specimens is a major asset. Th
e following procedures can be adopted for large throughput
laboratories with requirements of a high speed of turnaround.
22

1 Specimens continue to be sent to the laboratory in 10% buffered formalin, a necessary
precaution to avoid autolysis which may resul
t from delays and other mishaps that occur
during transportation of fresh specimens.

2 Following examination and sampling of these specimens, 2 mm thick blocks are placed in
cassettes, completely immersed in normal saline, irradiated to a temperature of 62

C and
held at this temperature for 30 seconds (this can be easily set in ovens which have
temperature probes). For convenience, 20 cassettes are placed in each of three beakers of
saline, equidistantly located at the periphery of the oven's rotating dish.

Although
morphological preservation is slightly better at higher temperatures, 62

C appears to allow
optimal preservation of tissue antigen.

3 Following irradiation, the tissue blocks are processed through cycles of absolute alcohol,
chloroform or xylene,

and wax in a vacuum
-
assisted automated processor.

Cycles of 1


hours and 3


hours are used, the former for endoscopic and other small
biopsies, and the latter for virtually all other tissue blocks. For convenience, an overnight
cycle of 16


hours can stil
l be used if required, details of such cycles being provided in
Table 1
. During the interim between microwave irradiation and commencement of tissue
processing, the tissue blocks can be held in 70% alcohol, Carnoy's solution, 10% buffered
formalin, or even normal saline. The first two solutions are particularly useful for tis
sues that
contain large quantities of fat.

The use of such a protocol allows the rapid preparation of good quality diagnostic sections
from most specimens. Large pieces of skin and dense tissue, such as myometrium require
immersion of approximately 30 minu
tes in formalin before microwave irradiation, a step
which can easily be introduced with little disruption to the procedure set out above.

MICROWAVE FIXATION OF BRAIN

Many of the brain enzymes that catalyse metabolism of compounds such as catecholamines,
a
cetylcholine, GABA, 5
-
hydroxytryptamine, cyclic AMP, and cyclic GMP are completely
and irreversibly decomposed by heat. Irradiation with MWs efficiently arrests such enzymatic
activity in brain tissue allowing for more accurate assays of these compounds an
d, at the
same time, hardening the tissues to allow easy dissection.

The irradiation of experimental brain tissue, previously perfused with physiologic saline or
formaldehyde has produced excellent tissue sections without the morphological changes that
re
sult from autolysis and dehydration and impregnation for routine paraffin processing.
23

Compared with cryostat sections of untreated brain, MW
-
irradiated tissue show fewer
changes from freezing
-
thawing and produce superior sections. Results are better in i
rradiated
tissue following perfusion with saline than those following perfusion with formaldehyde. In
addition, the Bodian stain and immunohistochemical staining of neurofilaments in axonal
structures are much more distinct in saline
-
perfused
-
irradiated ti
ssues.
23

MW TO ACCELERATE FIXATION

While MW irradiation may serve as the primary method of fixation, MWs can also accelerate
the fixing action (cross
-
linking) of aldehydes or alcohols. A three
-
step method using MW
irradiation to produce microscopic slices
of fresh human brain tissue within one working day
has been described.
24

The procedure comprises exposure of the whole brain, sprinkled with
physiologic saline, to MWs for 30 minutes, followed by irradiation of brain slices for 15
minutes before immersion
in 10% formalin for 3


hours. The final step is a period of MW
irradiation in formalin for six minutes.

MW irradiation can similarly be used to accelerate fixation of tissues in glutaraldehyde and
other cross
-
linking agents such as Karnovsky's fixative (0.
05% glutaraldehyde and 2%
formaldehyde) for electron microscopy (transmission and scanning). For example, tissue
samples immersed in 2

l of the aldehyde fixative can be fixed by irradiating to 50

C
(usually requiring 5
-
10 seconds). This is best achieved b
y locating the vial of fixative and
tissue in the centre 1.5 cm above the carousel on a polystyrene block as the irradiation is most
uniform at this site. After removal from the warm solution, the tissue is either stored in 0.1
mol/l sodium cacodylate buff
er with 0.02% sodium azide for up to 2 weeks, or immediately
processed.
25
-
26

After irradiation in 2.5% glutaraldehyde, the preservation of fine structural
features is similar to that of routinely processed tissues.
27

Additionally, renal biopsies fixed
usin
g MWs and prepared for both light and electron microscopy have been shown to be
suitable for immunofluorescence studies, with results equivalent to those of tissues directly
snap frozen.
28

The use of a MW
-
irradiation device that maximises the power output
and
coupling of the electromagnetic energy to the tissue sample, makes possible ultrafast MW
fixation with the process accomplished in as brief a period as 26 milliseconds in cross
-
linking
aldehyde solutions.
29

Although speed of turnaround is not as import
ant in the preparation of specimens for routine
electron microscopy as it is for light microscopy, the ultrastructural examination of fine
needle aspiration biopsies does require expedient processing. This aspirated sample is
irradiated in 8
-
10 ml of a 1%
glutaraldehyde
-
4% formaldehyde mixture in cacodylate buffer
for 25 seconds in a 650 Watt domestic oven. This is followed by dehydration, embedding in
epoxy resin and staining, with a section ready for examination within 2 hours.
30

MW
stimulated fixation fo
r electron microscopy has the added advantage of preserving of cellular
enzymes and proteins which are otherwise difficult to conserve with conventional fixation.
These include antigenic sites of rat muscle, chymase and other subcellular structures.
31
-
32

O
THER LABORATORY APPLICATIONS OF MICROWAVE IRRADIATION

An important application of MW irradiation is in the production of vastly improved cell form
and structure in cryostat sections. Immersion of freshly cut, frozen sections in Kryofix or
Wolman's solution

(95% ethanol and 5% glacial acetic acid) followed by exposure to MWs
for 15 seconds produces noticeable improvement in the quality of the microscopic image. The
cytomorphology is vastly superior to sections conventionally fixed in 95% ethanol, in 10%
buff
ered formalin, or in formalin vapour.

MW irradiation can also be used to accelerate almost every histochemical staining procedure
for light microscopy, staining of ultra
-
thin sections by uranyl

acetate and lead citrate for
transmission electron microscopy, immunohistochemical procedures, and all the stages of
tissue processing.
33
-
34

Excluded volume fixation

The addition of various polymers to a reaction mixture has the effect of 'fixing' small
d
iffusible molecules during the course of the reaction. As the rate at which such molecules
diffuse through the polymer is related to its size and concentration, the size of the molecule
which can diffuse through the polymer is progressively reduced as the
concentration of the
polymer rises. Twenty per cent polyvinyl alcohol or 20% polyethylene glycol (Carbowax
6000) have been used but mostly in an experimental situation.

Vapour fixation

Vapour as opposed to liquid fixation was originally used to retain solu
ble materials in situ by
converting them to insoluble products before contacting with water or non
-
aqueous solvents.
Various chemicals which act as vapour fixatives include aldehydes (formaldehyde,
glutaraldehyde and acrolein), osmium tetroxide, chromyl ch
loride, ethanol,
diethylpyrocarbonate, benzoquinone, and diacetyl; the most common being formaldehyde,
osmium tetroxide, and perhaps alcohol. The most important application of vapour fixation
has been the use of formaldehyde at elevated temperatures for th
e conversion of
catecholamines and 5
-
hydroxy
-
tryptamine in freeze
-
dried tissue to produce fluorescent
condensation products. This method
35

highlights the ability of highly reactive fixative vapour
to capture and render insoluble otherwise highly soluble, l
ow molecular weight compounds.
The usual source of monomeric formaldehyde is heated paraformaldehyde and the technique
can also be applied to frozen sections.

Osmium tetroxide at 37

C produces a vapour pressure which is sufficient to allow very
rapid pene
tration into freeze
-
dried blocks of tissue and exposure for 1 hour or less is usually
adequate.

Ethanol vapour at 60

C has a pronounced denaturing effect on freeze
-
dried tissues and
polysaccharides like glycogen become less soluble. There is, however, no
real application for
this technique in diagnostic pathology.

Freeze
-
drying

Stein et al
36

described a method of freeze drying at
-
45

C, 10
-
2 torr in the presence of
phosphorus pentoxide for up to 48 hours before embedding in paraffin, for the preservation o
f
labile lymphocyte antigens. While effective, the method is cumbersome and requires special
equipment. The resultant tissues have also proved to be difficult to section and often display
suboptimal morphology. Another method, freeze substitution using low

temperature plastic
embedding, avoids the need for fixation and retains tissue morphology and
immunoreactivity.
37

Postfixation

In situations where, for various reasons, the primary fixation process may not be optimal, the
wet tissue may be subjected to po
st fixation or secondary fixation. For example, tissues fixed
in buffered formalin may be subjected to further fixation with a mercuric chloride
-
formaldehyde solution for a period of several hours to improve staining of sections. Other
post fixatives such
as a mercuric chloride
-
picric acid
-
formaldehyde mixture may be used.
The main disadvantages of post fixation is the extra time and costs involved as well as the
toxicity of the mercuric solution.

Post fixation in osmium tetroxide is common in electron micr
oscopy for tissue blocks
previously fixed in glutaraldehyde. Osmium tetroxide improves the staining of cell
membranes although the actual mechanism involved is not completely known.

Fixation mixtures

A mixture of osmium tetroxide and glutaraldehyde has bee
n shown to be useful for neutral fat
and fine structural localisation of acid phosphatase. Other mixtures such as osmium tetroxide
-
zinc iodide post fixation have been used for delineating synaptic vesicles and Langerhans'
cells, and glutaraldehyde and carb
odiimide have been used as a primary fixative for electron
microscopic immunohistochemistry. McDowell and Trump
38

developed a general purpose
fixative for both light and electron microscopy comprising a mixture of formaldehyde and
glutaraldehyde.

Additives

Tannic acid

Tannic acid is a useful addition to the fixation solution as it precipitates of a number of
polypeptides and proteins. Tannic acid penetrates tissue easily, imparting high contrast to
membranes and staining amorphous material and elastic fibre
s. It appears to act as a mordant
for heavy metal staining and prevents the loss of certain tissue components. Its action is
independent of aldehyde groups in the tissues and its mordanting function is dependent on
carboxyl and at least one hydroxyl on the

benzene ring. Tannic acid has been added to
formaldehyde
-
glutaraldehyde solutions, allowing endogenous lactoperoxidase in mammary
tissue to be demonstrated, and it may also increase the retention of lipids in tissues. Tannic
acid diffuses easily through t
issues, penetrating and enhancing the density of damaged cells
allowing their differentiation from intact cells both by light and electron microscopy.

Phenol

The addition of 2% phenol has an accelerating effect on neutral buffered 4% formaldehyde as
a fixative with tissue sections showing improved nuclear and cytoplasmic detail, reduced
shrinkage and distortion and an absence of formalin pigment.
39

Resin
-
embedd
ed tissues fixed
in the phenol
-
formaldehyde fixative gave satisfactory preservation of ultrastructural features.

Heavy metal solutions

Transitional metal salts such as zinc can be potent protein precipitants, forming insoluble
complexes with polypeptides.
Zinc formalin has been proposed therefore as a fixative to
enhance immunostaining
40

and antigen preservation.
41

There is some suggestion that post
fixation in zinc sulphate may also improve immunostaining.
42

The introduction of zinc
formalin (1% zinc sulph
ate in 3.7% unbuffered formalin) into automated tissue processors
has produced no deleterious effects or damage.
41

Lanthanum

Lanthanum in its colloidal form has been added to the primary fixative to demonstrate
intercellular spaces and cell junctions. It has also been used with Alcian Blue in fixation to
demonstrate acid muco
-
substances on cell surfaces and the addition of lanth
anum to
glutaraldehyde will prevent the formation of lacunar spaces around chondrocytes in pre
-
mineralised cartilage, probably by binding to and fixing negatively charged molecules.

Lithium

Lithium salts have been used to combat volume changes, for exampl
e, pre
-
treatment of
glutaraldehyde
-
fixed tissues with isotonic lithium reduces the shrinkage brought about by
ethanol dehydration.

Detergents

Detergents have been used with the primary fixative to enhance the effect of some other
reagent used simultaneousl
y or subsequently in the overall technique. For example saponin
and Novidet aid the subsequent use of antibodies whilst Triton X
-
100 increases tissue
permeability for Ruthenium Red. It should be noted that detergent generally has a deleterious
effect on cy
tological detail and its application is limited to only specific situations.

Factors involved in fixation

Temperature

While the fixation of specimens for standard histology is generally carried out at room
temperature for convenience, for electron microsco
py and some histochemical procedures,
the temperature for fixation is usually 0
-
4

C. At this lower temperature range autolysis is
slowed down, as is the diffusion of various cellular components, allowing a more life
-
like
appearance of the tissues. However
chemical reactions, including those involved in the
fixation process are frequently more rapid at higher temperatures. The use of heat for fixation
in microbiology and for blood films is well known and the heating of formalin to
temperatures of 60
-
70

C can

be used for the rapid fixation of very urgent biopsy specimens,
although the risk of tissue distortion is increased.

Size of specimens and penetration of fixative

The penetration of fixatives into tissue is a relatively slow process and tissue blocks shou
ld
either be small or thin, in order to obtain satisfactory fixation. Large specimens should be
opened and washed of contents or sliced thinly before placement in fixative. The penetration
of fixatives into tissues can be determined from the following equa
tion based on the laws of
diffusion:

d = K


t

Where

d is the depth penetrated

t represents time

K (a constant) is the coefficient of diffusibility

of the fixative and is specific for each fixative.
It represents the distance in millimetres the fixative has diffused into the tissue at 1 hour.

An indication of the relative diffusibility of various fixatives in a uniform tissue such as the
liver, at ro
om temperature are given in
Table 2
. More recent work suggests that in human
tissues, the rate of fixative diffusion may even be lower.
43

Changes in volume

The mechanisms involved in volume changes

in tissues with fixation are not well understood.
Those suggested include inhibition of respiration, changes in membrane permeability and
changes in ion transport through membranes. Some intercellular substances such as collagen
may swell when they are fi
xed. There are also changes in volume which occur subsequently
during dehydration and paraffin embedding. It has been estimated that tissue fixed in
formaldehyde and embedded in paraffin wax shrinks by 33% and this is evident when
paraffin embedded section
s are compared with frozen sections, the latter showing larger
nuclei and cells.

pH and buffers

The hydrogen ion concentration varies between fixatives, but in general, the pH should be
kept in the physiological range, between pH 6
-
8. This can be maintaine
d by buffer systems,
the most common being phosphate,
s
-
collidine, bicarbonate, Tris, veronal acetate and
cacodylate. The chosen buffer should not react with the fixative as this will reduce both the
buffering power and the function of the fixative. Furthe
rmore, the buffer should not inhibit
enzymes or react with the incubation medium if histochemical procedures are to be
performed.

Osmolality

The addition of a buffer to the fixative solution may alter the osmotic pressure exerted by the
solution. Hypertoni
c solutions give rise to cell shrinkage whereas isotonic and hypotonic
fixatives result in cell swelling and poor fixation. With electron microscopy, the best results
are obtained using slightly hypertonic solutions (isotonic solutions being 340 mOsm)
adju
sted using sucrose.

Concentration of fixatives

Some fixatives are effective within a range of different concentrations, for example,
glutaraldehyde which may be used as a 4% solution is effective as low as 0.25%, provided
the pH is maintained in the physio
logical range. This allows large volumes of aldehyde
fixatives to be prepared at one time. (The presence of a buffer may cause polymerisation of
the aldehyde with a consequent decrease in its concentration). There is, however, some
variation in the stainin
g intensity of tissues with variation in the concentration of fixative.

When vehicles other than buffer and water are added the effectiveness of the fixative solution
may be altered. For example, some salts have denaturing effects while others such as
ammo
nium sulphate and potassium dihydrogen phosphate strongly stabilise proteins. Sodium
chloride and sodium sulphate are used in various fixative mixtures containing mercuric
chloride because sodium chloride may increase the binding of mercuric chloride to th
e amino
groups of proteins and may also dissolve coagulated proteins.

Duration of fixation

It is common practice to fix 2 mm thick tissue blocks in buffered formalin for 4
-
8 hours,
possibly followed by a period in formol

sublimate. Large specimens and viscera are cut into 5
mm slices or viscera are emptied and pinned out on a board, before fixing overnight in
buffered formalin. This allows easier handling, examination and dissection, particularly for
the sampling of lymph

nodes. In the case of electron microscopy, diced tissues are fixed for 3
hours in glutaraldehyde before placing in a holding buffer such as sodium cacodylate.

There is evidence that prolonged fixation in aldehydes can cause shrinkage and hardening of
tiss
ue and severe inhibition of enzyme activity. Prolonged fixation with oxidising fixatives
can degrade tissues by oxidative cleavage of proteins and loss of peptides.

Fixation of specific substances

Glycogen

A variety of glycogens occur naturally and show d
ifferent degrees of polymerisation. The less
highly polymerised forms are not well fixed by routine fixatives and diffuse into the fixing
fluid. This occurs in cases of glycogen storage disease where glycogen is predominantly of
the lighter type. In contra
st, the larger molecules of more highly polymerised glycogens are
retained with a wide variety of fixatives as well as alcohol
-
containing reagents. The retention
of glycogen is thought to be the result of trapping in a matrix or mesh of fixed protein, or d
ue
to its covalent binding to protein which renders it insoluble in water. However, there appears
to be stronger support for the concept that removal, by dehydration, of bound water
molecules from normal forms of tissue glycogen decreases solubility, amoun
ting to
denaturation.

The use of alcohols has, therefore, been the main method of fixing glycogen in tissues.
Earlier fixatives included ice
-
cold picro
-
alcohol
-
formalin or cold alcohol or a mixture of 96%
alcohol saturated with picric acid, 40% formalin,
and acetic acid. Chemical assays on rat liver
have shown 100% ethanol to be clearly superior for the fixation of glycogen. Bouin's fixative
is also a useful fixative for glycogen.

Lipids

With standard methods of fixation, lipids are largely lost from tissu
es during processing and
only two reagents fix lipids in the true sense of rendering them insoluble. These are osmium
tetroxide and chromic acid, both of which alter the chemical reactivity of the lipid
considerably. While several fixatives will preserve l
ipids, they generally do not alter their
solubility in the lipid solvents used in tissue processing. Baker's fixative, designed for the
preservation of phospholipids, uses formalin together with calcium and cadmium chlorides
(the last, being expensive, has

subsequently been replaced by cobalt nitrate). While
phospholipids are preserved, they are not prevented from diffusing into the fixing fluid and
are still removable by fat solvents. Lipids can be demonstrated in cryostat sections fixed with
reagents cont
aining mercuric chloride and potassium dichromate such as Elftman's fluid, with
fixation for unsaturated lipids completed over 3 days at room temperature.

Various additives have been mixed with glutaraldehyde in order to demonstrate lipids in
electron micr
oscopy. Digitonin added to glutaraldehyde preserves cholesterol
44
-
45

and
Malachite Green included with glutaraldehyde or Karnovsky's fixative retains various lipids
such as phospholipids, fatty acids, glycolipids and choline plasmalogen.
46

Imidazole
introd
uced into the post osmication of glutaraldehyde
-
fixed tissue demonstrates unsaturated
fatty acids and phospholipids as electron
-
dense deposits.
47

Proteins

The fixation of tissue proteins by aldehydes is largely through production of cross
-
linkages
between
various reactive groups in proteins. Most fixatives preserve proteins adequately in 1
to 2 days. Glutaraldehyde fixes proteins very rapidly whereas formaldehyde reacts reversibly
over the first 24 hours. Osmium tetroxide reacts with proteins by producing c
ross
-
links and
protein gels. Prolonged exposure to osmium tetroxide causes the breakdown of proteins.

Mucosubstances

Among the mucosubstances are the single component polysaccharides such as glucose, starch
and cellulose which are referred to as homoglycan
s whereas those with two or more
monosaccharide components are the heteroglycans. The latter are composed of the
glycosaminoglycans such as keratosulphate and sialoglycans, and the
glycosaminoglucoronoglycans comprising hyaluronic acid, chondroitin sulphat
es and
heparin. Protein
-
polysaccharide complexes are known as proteoglycans.

The loss of mucosubstances from tissue during fixation is well recognised and many fixatives
have been suggested to prevent this. Four per cent basic lead acetate was introduced a
s a
fixative for acid heteroglycans and 1% lead nitrate in place of acetate has also been used.
Alcoholic 8% lead nitrate, with or without 10% formalin, has been employed for connective
tissue glycosaminoglucoronoglycans. Formalin
-
alcohol mixtures have als
o been used and
calcium acetate has been added to formalin as a cationic precipitate for acidic mucins.
Formalin has always been an essential component of whatever fixative used to ensure the
preservation of proteoglycans, however, an appreciable proportio
n of tissue hetero
-

and
proteoglycans remains soluble unless subject to further precipitation in 70
-
80% ethanol (for
3
-
6 days) before clearing and embedding in paraffin.

The reactions of cetylpyridinium salts (with acid glycosaminoglycans) and acridines to

form
insoluble complexes are superior to formalin, Carnoy's solution and formalin
-
alcohol
mixtures, for the preservation of heteroglycans. The most successful method for preserving
all types of mucin is freeze drying followed by hot formaldehyde vapour.

V
arious cationic dyes have been introduced in attempts to preserve glycosaminoglycans for
ultrastructural analysis. Toluidine Blue, Saffranin O, Acridine Orange and more recently a
phthalocyanin
-
like dye, Cuprolinic Blue
48

preserve and stain proteoglycans w
ithout fixation
by glutaraldehyde or formaldehyde. The reaction is based on electrostatic attraction between
the positive dye and the polyanionic glycosaminoglycan component.

Nucleic acids and nucleoproteins

The nucleic acids exist in many different states

of polymerisation and any method of fixation
induces changes in their physical state. Formalin is not a particularly good fixative for nucleic
acids and nucleoproteins as it blocks a large number of reactive groups reducing their
subsequent staining by bo
th basic and acid dyes. This can be improved by adding mercury or
chromium salts. Precipitant fixatives like alcohol, acetic acid, and Carnoy's fluid are
preferable since these agents precipitate nuclear proteins and at the same time progressively
break th
e bonds between nucleic acids and proteins, thereby increasing the number of acid
groups available for staining. However, prolonged fixation in acid fixatives such as Carnoy's
reagent profoundly alters nuclear proteins and extracts RNA and DNA.

Biogenic am
ines

The biogenic amines include two main groups, the catecholamines, adrenalin and
noradrenaline, and the indolalkylamines, dopamine, DOPA and 5
-
hydroxytryptamine. The
chromaffin reaction may be demonstrated by fixing the tissue in a solution of formalin
with
sodium acetate and potassium dichromate. The biogenic amines can also be demonstrated by
formaldehyde
-
induced fluorescence.

Glutaraldehyde at pH 7.2
-

7.4 precipitates noradrenaline but adrenalin dissolves into the
solution unless 1.5% of potassium d
ichromate is added. A more effective method of retaining
biogenic amines for ultrastructural examination is to use a three
-
stage fixation procedure.
49

This comprises primary fixation in a mixture of 1% glutaraldehyde, 0.4% formaldehyde,
sodium chromate and

potassium dichromate followed by storage for 18 hours in a mixture of
sodium chromate and potassium dichromate, and finally, post fixation in 2% osmium
tetroxide, sodium chlorate and potassium dichromate.

Enzymes

Enzyme activity is best demonstrated histo
chemically in fresh frozen sections. The most
common methods of preserving enzymes for paraffin embedding are fixation in alcohol or
acetone usually at 4

C. Alkaline phosphatase

activity is retained by both these fixatives
when used cold, and the fixing capability of alcohol improves when saturated with sodium


-
glycerophosphate. Subsequent clearing of alcohol or acetone
-
fixed blocks in light petroleum
(petroleum ether) and embe
dding in vacuo in low melting point (42
-
44

C) paraffin
improves enzyme preservation further.

The most significant problem in enzyme histochemistry

is false localisation due to diffusion
of the enzyme. To this end, various fixatives have been described for the optimal preservation
of specific enzymes. Formalin
-
sucrose
-
ammonia and 1
-
4% glutaraldehyde have been used for
cholinesterases.
3,50

The fixatio
n of acid phosphatase can be achieved with formaldehyde and
formalin containing 0.1% chloral hydrate will preserve ß
-
glucuronidase.

Agonal changes and fixation artefacts

Ideally, tissues should be fixed immediately and completely from the living state, but

this
cannot be achieved for human tissues. As most tissues are removed surgically, the organ or
tissue is relatively anoxic for some period because of anaesthesia and the placement of
surgical clamps and ligatures to stop bleeding. Furthermore, when the t
issue is placed in
fixative, there is a latent period before adequate penetration of the tissue. Anoxic changes
including damage to mitochondria are noticeable ultrastructurally within 10 minutes and
enzymes such as those concerned with oxidative phosphory
lation are lost within an hour.
There is also variation within the tissue as cells in the centre of the block suffer more anoxia
due to delays in penetration of the fixing solution. Anoxic changes occur more rapidly at
room temperature than in the cold and

thus are exaggerated with post mortem material as the
body remains at room temperature for variable periods before it is transferred to the mortuary
and refrigerated. Furthermore, as the autopsy may not be conducted for several hours or days
after death,
some degree of autolysis invariably occurs.

While one of the major aims of fixation is preservation of tissues in as life
-
like state as
possible, it is important to appreciate that fixation itself may cause certain artefacts.
Expansion and shrinkage of tis
sues during fixation have been previously discussed. Another
important artefact relates to the movement of unfixed material so that organelles and other
subcellular structures may be falsely localised at sites where they do not belong. For example,
the ves
icles which are commonly seen fused with the fibroblast cell membrane are not seen
with freeze fracture techniques, and it is felt that the vesicles exist in the subjacent cytoplasm
and are induced to fuse with the cell membrane by glutaraldehyde fixation.

Glutaraldehyde
preserves the interdigitations of cell membranes whereas osmium tetroxide causes their
breakdown into vesicles. It has been noted that tight junctions in rat liver and small intestine
vary according to the method of preparation, and tight j
unction fibrils are produced by the
cross
-
linking of junctional proteins by glutaraldehyde. Unfixed haemoglobulin diffuses from
red blood cells to the periphery of a block because of the relatively slow penetration of
glutaraldehyde. This illustrates the a
bility of large molecules to diffuse during fixation and
also underlines the necessity to use only small blocks of tissue.

Not only may large molecules move within a block but materials may diffuse from the tissue
altogether. During fixation this occurs wi
th both large and small molecules including
inorganic ions and cofactors for enzymes. Biogenic amines, for example, are stored in
membrane
-
bound granules in the cells of the adrenal medulla, neurons and other
neuroendocrine organs. Fixation denatures chrom
ogranin, an associated protein, with the
release of the biogenic amines and ATP. Unless the amines can be fixed in some way such as
by precipitation, they may be lost from the tissue. Indeed, this diffusion can be seen
macroscopically when adrenal tissue i
s placed in iodate. As catecholamines diffuse from the
tissue into the fixing solution, they react with iodate and can be seen as a haze of red
aminochromes. The opposite situation may also occur with false fixation of extraneous
material within the tissue
. This is particularly so when employing radioactive labelled amino
acids, sugars, thymidine and uridine.

Chemical changes caused by fixation may give false histochemical reactions in the tissue. For
example, glutaraldehyde will add carbonyl groups to tiss
ues and these will react with Schiff's
reagent. In removing excess mercuric chloride after fixation, substances such as histidine,
tyrosine and mercaptides may also be removed.

Lastly, if the fixative employed was chosen for the preservation of a specific
substance, it is
likely that many other cellular substances will be lost as there is currently no single fixative
that will preserve all tissue substances. The loss of components may be due to their lack of
reaction with a fixative and subsequent removal d
uring processing or may result from
degradation by the fixative as in the case of small molecules such as biogenic amines; while
glutaraldehyde will precipitate noradrenaline, adrenalin is lost from the tissues.
Formaldehyde at pH 7 causes the loss of abou
t 60% of catecholamines from chromaffin
granules and 5
-
hydroxytryptamine is lost during fixation in formaldehyde and
glutaraldehyde51. Formalin is an inadequate fixative for the proteoglycans of many pituitary
glands and many neuropeptides such as luteiniz
ing hormone
-
releasing hormones are ethanol
soluble and may be lost during dehydration. The difficulty in retaining lipids in sections is
well recognised and the loss of mucosubstances during fixation is also well documented.
Enzymes may be released during
fixation and ions may be lost from tissues. Prolonged
fixation in formaldehyde results in the loss of water soluble materials particularly when
fixation exceeds 6 hours. Proteins are degraded by osmium tetroxide.

Fixatives: a summary

It is clear that there

is no universal fixative which will serve all requirements. Each fixative
has specific properties and disadvantages and their many different effects emphasise the
necessity for careful consideration and selection of the appropriate fixing reagent when
stu
dies of specific cellular substances are planned. Ten per cent buffered formalin and 2.5%
glutaraldehyde are currently the most widely used fixatives for routine light microscopy and
ultrastructural studies, but they too, have inherent disadvantages which
the user should be
well conversant with. Increasing interest in tissue and cell constituents including cellular
proteins detectable by immunohistochemical techniques, imposes additional requirements for
the preservation of such substances. Microwave irradi
ation is a physical modality which
provides an alternative method of primary fixation. It is rapid, cheap, safe and clean and can
also be employed as a means of stimulating accelerated fixation by other known fixatives and
mixtures.

Formulations for variou
s fixtives

The details provided relate to commonly used fixatives. Many variations are available and
more specialised fixative solutions are not provided.

Formaldehyde solutions

10% neutral buffer formalin (4% formaldehyde)

REAGENTS REQUIRED

1 40% formalde
hyde 100 ml

2 Distilled water 900 ml

3 Sodium dihydrogen orthophosphate 4 g

4 Disodium hydrogen orthophosphate (anhydrous) 6.5 g

METHOD

Prepare, using quantities indicated. Fixation time: 24
-
72 hours.

Baker's formol
-
calcium (modified)

REAGENTS REQUIRED

1
40% formaldehyde 100 ml

2 Distilled water 900 ml

3 10% calcium chloride 100 ml

4 7 g of cadmium chloride is sometimes added to the mixture

METHOD

Prepare, using quantities indicated. Fixation time: 16
-
24 hours.

Formol saline

REAGENTS REQUIRED

1 40% formald
ehyde 100 ml

2 Sodium chloride 9 g

3 Tap water 900 ml

METHOD

Prepare, using quantities indicated.

Alcoholic formaldehyde

REAGENTS REQUIRED

1 40% formaldehyde 100 ml

2 95% alcohol 900 ml

3 0.5 g calcium acetate may be added to this mixture to ensure neutral
ity

METHOD

Prepare, using quantities indicated. Fixation time: 16
-
24 hours.

Paraformaldehyde

REAGENTS REQUIRED

1 Solution A

2.26% sodium dihydrogen orthophosphate 41.5 ml

2.52% sodium hydroxide 8.5 ml

Heat to 60

C
-
80

C in a covered container

2 Paraformalde
hyde 2 g

METHOD

1 Add paraformaldehyde to solution A, stirring until the mixture is clear.

2 Filter and cool. Adjust pH to 7.2
-

7.4.

3 Prepare fresh for use (duration of fixation depends on size of specimen and whether for
light or electron microscopy).

B
uffered formaldehyde
-
glutaraldehyde 200 mOsm
38

REAGENTS REQUIRED

1 Sodium dihydrogen orthophosphate 1.6 g

2 Sodium hydroxide 0.27 g

3 Distilled water 88 ml

4 40% formaldehyde 10 ml

5 50% glutaraldehyde 2 ml

METHOD

Prepare, using quantities indicated. Fixat
ion time: 16
-
24 hours.

Alcoholic fixatives

Carnoy's fixative

REAGENTS REQUIRED

1 Absolute ethanol 60 ml

2 Chloroform 30 ml

3 Glacial acetic acid 10 ml

METHOD

Prepare, using quantities indicated. Fixation time: 1
-
5 minutes.

Methacarn

REAGENTS REQUIRED

1 Abs
olute methanol 60 ml

2 Chloroform 30 ml

3 Glacial acetic acid 10 ml

METHOD

Prepare, using quantities indicated. Fixation time: 5
-
6 hours.

Wolman's solution

REAGENTS REQUIRED

1 Absolute ethanol 95 ml

2 Glacial acetic acid 5 ml

METHOD

Immerse frozen section
in solution and microwave at 650 watts for 15 seconds.

Acetic alcohol formalin

REAGENTS REQUIRED

1 40% formaldehyde 10 ml

2 Acetic acid 5 ml

3 Ethanol 85 ml

METHOD

Prepare, using quantities indicated. Fixation time: 24 hours at 4

C.

Picric acid fixatives

Rossman's fluid

REAGENTS REQUIRED

1 100% ethanol saturated with picric acid 90 ml

2 Neutralised commercial formalin 10 ml

METHOD

Prepare, using quantities indicated. Fix for 12
-
24 hours and wash very well in 95% ethanol.

Gendre's fluid

REAGENTS REQUIRED

1
90% ethanol saturated with picric acid 80 ml

2 40% formaldehyde 15 ml

3 Glacial acetic acid 5 ml

METHOD

Prepare, using quantities indicated. Fixation is normally for 4 hours, followed by washing in
80%, 95% and 100% ethanol.

Bouin's fluid

REAGENTS REQUIRED

1 Saturated aqueous picric acid solution 75 ml

2 40% formaldehyde 25 ml

3 Glacial acetic acid 5 ml

METHOD

1 Prepare, using quantities indicated. Fixation may vary from a few hours to 18 hours.

2 Washing with 70% ethanol after fixation will remove most of
the yellow colour. Sections
can also be washed after removal of paraffin wax.

Mercuric fixatives

Buffered formaldehyde sublimate

REAGENTS REQUIRED

1 Mercuric chloride 6 g

2 Distilled water 90 ml

3 Sodium acetate 1.25 g

4 40% formaldehyde 10 ml

METHOD

Prepa
re, using quantities indicated. Fixation time: 16
-
18 hours.

Zenker's fluid

REAGENTS REQUIRED

1 Distilled water 950 ml

2 Potassium dichromate 25 g

3 Mercuric chloride 50 g

4 Glacial acetic acid 50 g

METHOD

Prepare, using quantities indicated. Fixation is no
rmally for 4
-
24 hours followed by an
overnight wash.

Helly's fluid

REAGENTS REQUIRED

1 Solution A

Distilled water 1000 ml

Potassium dichromate 25 g

Sodium sulphate 10 g

Mercuric chloride 50 g

2 Solution B

40% formaldehyde 50 ml

METHOD

Add solution A to sol
ution B immediately before use.

B5 fixative

REAGENTS REQUIRED

1 Stock reagent A

Mercuric chloride 60 g

Sodium acetate 12.5 g

Distilled water l

2 Stock reagent B

10% buffered neutral formalin

METHOD

To prepare a working solution mix 90 ml stock reagent A wi
th 10 ml stock reagent B.
Fixation time: 5
-
8 hours.

Susa fluid

REAGENTS REQUIRED

1 Distilled water 80 ml

2 40% formaldehyde 20 ml

3 Glacial acetic acid 4 ml

4 Trichloroacetic acid 2 g

5 Mercuric chloride 4.5 g

6 Sodium chloride 0.5 g

METHOD

Prepare, using
quantities indicated. Fixation time: 12 hours.

TECHNICAL NOTES

Mercuric chloride is usually combined with other fixatives as it penetrates poorly and
produces tissue shrinkage (formaldehyde is the additional fixative in the above mixture).
Fixatives contai
ning mercuric chloride produce a black precipitate of mercury which can be
removed by placing sections in 0.5% iodine solution in 70% ethanol for 5
-
10 minutes,
followed by rinsing in water, decolourisation in 5% sodium thiosulphate for 5 minutes and
rinsin
g in water again.

Fixation of specific substances

Glycogen

Alcoholic fixatives such as Rossman's fluid, Gendre fluid and ethanol fix glycogen.

LIPIDS

Elftman's fixative

REAGENTS REQUIRED

1 Mercuric chloride 5 g

2 Potassium dichromate 2.5 g

3 Water 100 ml

M
ETHOD

Prepare using quantities indicated. Fixation time: 3 days at room temperature.

Swank and Davenport's fixative

REAGENTS REQUIRED

1 1% potassium chlorate 60 ml

2 40% formaldehyde 12 ml

3 Acetic acid 1 ml

METHOD

Prepare, using quantities indicated.
Fixation time: 6
-
10 days.

Other fixatives for lipids include Baker's formol
-
calcium for phospholipids, and osmium
tetroxide for unsaturated lipids.

MUCOSUBSTANCES

Lillie's alcoholic lead nitrate

REAGENTS REQUIRED

1 Lead nitrate 8 g

2 40% formaldehyde 10 ml

3 Water 10 ml

4 Ethanol 80 ml

METHOD

Prepare, using quantities indicated. Fixation time: 24 hours at room temperature.

Cetylpyridinium chloride (C.P.C.)

REAGENTS REQUIRED

1 40% formaldehyde 10 ml

2 Cetylpyridinium chloride 0.5 g

3 Water 90 ml

METHOD

Prepa
re, using quantities indicated. Fixation time: 48 hours.

Lead subacetate
-
ethanol fixative

REAGENTS REQUIRED

1 Lead subacetate 1 g

2 Ethanol 50 ml

3 Water 50 ml

4 Acetic acid 0.5 ml

METHOD

Prepare, using quantities indicated. Fixation time: 24 hours.

BIOGE
NIC AMINES

Potassium dichromate
-
acetate
-
formaldehyde fixative

REAGENTS REQUIRED

1 5% potassium dichromate solution 60 ml

2 1 mol/l sodium acetate 10 ml

3 40% formaldehyde 12 ml

4 Distilled water 18 ml

METHOD

Prepare, using quantities indicated. Fixation ti
me: 18 hours.

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