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Abstract
Acrylic resin mixtures are now widely used as embedding
media for the preparation of tissue sections. Most of these
mixtures are based on 2-hydroxyethyl methacrylate (glycol
methacrylate, GMA). Resin embedding preserves tissue
components far better than paraffin, celloidin or frozen sections. The present review describes the basic principles and
trouble shooting, in particular: the chemical and physical
properties of GMA, and components used for GMA mixtures; fixation of tissues for resin embedding; methods for
dehydration; microtomy; stretching on water and mounting
in relation to the final dimensions of GMA sections; staining of GMA 3embedded tissue sections; and the use of GMA
resins in immunohistochemistry. In addition, standard, step
by step procedures for embedding tissues in GMA is included. (The J Histoteclzaol 19:297-3 11, 1996)
Key words: acrylic resins, embedding, glycol methacrylate,
immunohistochemistry, tissue processing
Introduction
In order to obtain thin sections of biological specimens,
the embedding media paraffin and celloidin were introduced
into light microscopy by Klebs and Duval (1,2). Paraffin
and celloidin mechanically support fragile tissue components during sectioning, thus preventing distortions. Selective staining of tissues and cellular elements in sections then
enables precise investigation of microscopical structures to
be carried out. Resin embedding media commonly used in
electron microscopy preserve morphology far better than do
paraffin and celloidin. A diversity of methacrylate, polyester, and epoxide resins found application in electron microscopy. At first polymers such as polymethylmethacrylate
Shrinkage of tissues after GMA embedding is less pronounced than with paraffin (12,13,15,16). Resin sections vary in thickness much less than do paraffin sec-
297
Disadvantages
Apart from these advantages, GMA ernbedding techniques have also their limitations and disadvantages:
Several components of typical resin mixtures are more
or less toxic. Thus, not only self-prepared mixtures but
also comlnercial embedding kits must be handled with
care.
Since GMA monomer is water-miscible. it is sometimes used to dehydrate tissues prior to embedding
(32). However, bear in mind that GMA can extract
almost all neutral lipids (33). Embedding in the cold
reduces such lipid extraction (34).
Embedding large tissue blocks in GMA may be difficult and requires special procedures (35). Knowledge
of the com~ositionof the resin mixtures in use. and
information' concerning the possibility of modifying
the concentrations of the components, is required. At
this moment a suitable com~nercialGMA mixture to
embed large tissue blocks is not yet available.
sections have been a logical consequence of the developments of resin embedding techniques in light microscopy.
Doubtless such innovations will continue; indeed the special
features of this embedding lnediurn almost guarantees an
accelerating interest by ever increasing numbers of workers.
'42 H2
HO-C - C -OH
ETHYLENE GLYCOL
,CH3
P
,
H2C=C
METHACRYLIC ACID
'c
\ OH
/ CH3
H2C = C\ (0
C
'0-
GLYCOL METHACRYLATE
H2
C-C
H2
-OH
Storage
Hydroxyalkyl methacrylates such as GMA need to be
properly stored in order to maintain stability. To avoid polymerization, pure GMA has to be kept below 30C and in
the dark. GMA-monomer must contain about 20 ppm of
dissolved oxygen. At higher temperatures inhibitors such as
hydroquinone and hydroquinone mono-methylether, varying in concentration from 50 to 400 ppm, are necessary to
prevent spontaneous polymerization of GMA during storage, and adequate storage is guaranteed only in the presence
of atmospheric oxygen. Consequently, bottles and containers must be filled only to a maximum of 85% with GMA.
Spontaneous development of benzoyl monomeric, and
oligomeric radicals in initiated GMA solutions leads to decrease of inhibitor concentrations (45). Consequently the
shelf-life of GMA solutions is shortened.
Plasticizers
Plasticizers are small molecules added to resins to improve their flexibility and processability. In light microscopy external plasticizers (plasticizers which do not copolymerize) are mostly used. The characteristic of
plasticizers is that they lower the modulus of elasticity and
the glass transition temperature (Tg) of polymers, but do not
alter the chemical nature of the final polymer. The Tg can be
defined as the temperature at which glassy amorphous polymers become flexible or rubber-like because of the onset of
segmental motion (free rotation of the covalent bonds). Below their Tg thermoplastic resins are in a relative brittle
state and in consequence difficult to cut. The Tg of GMA is
55C. Addition of plasticizers may shift down the T,0 considerably, relegating brittleness to a lower temperature. A
clear linearity exists between the Tg and the amount of a
plasticizer. For cutting resin blocks the "use" temperature
must be above Tg. Resin embedding nlixtures are composed
The Journal of Histotechnology I Vol. 19, No. 4 1 December 1996
100 ml
1 gm
Enzbeddiizg Solution
Infiltration Solution A
Hardener I1 (contains the accelerator solution,
a barbituric acid derivative)
15 ml
1 ml
90 ml
(monomer)
10 ml
0.4 ml
(plasticizer)
(crosslinker)
0.6 gm
(initiator)
1 part
30 parts
by volume
Solution C (Embedding
Solution)
Solution A:Solution B (vlv),
varied from 30: 1 to 40: 1
Zizitiator-Accelerator Systeins
Barbitiiric Acid Derivatives in Coinbination with
Clzloride Ions
The work of Bredereck et a1 describes this less toxic
initiator-accelerator system (52). It is based on the principle
that compounds with the general formula A-CH(R)-B
(where A and B indicate electron attracting groups, R a H
atom or an alkyl group) initiate the polymerization of monomers like methacrylates in the presence of Cu++ (eg, provided by copper(II)acetylacetonate), C1- (eg, dibutylaminehydrochloride or dila~~ryldimethyl
ammonium chloride),
and oxygen or peroxides. The = CHR group may be part of
a carbo- or hetero-cyclic ring such as barbituric acid. The
start of polymerization is explained as an autoxidation of the
active CH-group to the hydroperoxide. We have modified a
simplified scheme of the chemistry of this barbituric acid
derivative-chloride ion initiator system, shown in Figure 2
(53).
Dibeizzoyl Peroxide in Cornbiizntion ~vitlzn Tertiary A~nirze
This system is a representative of the peroxide-tertiary
aromatic amine, initiator-accelerator system (Figure 3).
Normally dibenzoyl peroxide (BPO) decomposes at 70C,
producing free radicals, which can be used for the initiation
of the polymerization of GMA. However, this relatively
high processing temperature is harmful to delicate tissue
structures. This can be avoided by using accelerators. At
room temperature the decomposition of dibenzoyl peroxide,
which produces free radicals, can be accelerated by tertiary
amines such as N,N-dimethylaniline (DA). The mechanism
of acceleration of peroxide-initiated polymerization by ter-
Exothermic Polyinerizatio~zReaction
The polymerization of GMA is exothermic (50 kJImol) and
temperatures in excess of 100C can easily be exceeded in the
center of the polymerizing resin when uncontrolled amounts of
initiators and accelerators are used. Possibilities of regulating
heat production during room temperature polymerization were
investigated by Gemts and van Leeuwen (49). The polymerization of GMA using dibenzoyl peroxide-tertiary aromatic
amine as initiator-accelerator system was studied under standardized conditions; in a climate controlled room at 20"C, with
a relative humidity of 60%. In particular the influences of BPO
and inhibitor concentrations and the ambient temperature upon
the maximum temperature reached during polymerization
were studied. These experiments indicated that the peroxidetertiary aromatic amine, initiator-accelerator system is very
sensitive to variations in the concentrations of the chemicals
used and to fluctuations of ambient temperature. GMA mixtures containing low concentrations of BPO yield low maximum temperatures during polymerization. However, too low a
concentration of BPO is often associated with incomplete polymerization, or even with no polymerization at all. Too high
a concentration of BPO is associated with undesirable high
temperatures within the resin block. Variations in the concentration of the accelerator may also influence the maximum
temperature and the moment when the maximum temperature
is reached.
Furthermore, factors external to the polymerization mixture can also influence polymerization. The use of appropriate heat conducting blockholders proved to be an essential temperature-decreasing factor in the embedding
procedure. In addition the total volume of GMA monomer
used also correlates with the maximum temperature
reached. Ashford et a1 reported that even polymerization of
GMA at lower temperatures (<OC) does not allow total
dissipation of the heat of polymerization (56).
We also investigated the polymerization characteristics of
the initiator-accelerator system making use of barbituric
acid derivatives in combination with chloride ions (eg,
Technovit 7100 and HistoResin) (51). We found that this
system behaves quite differently from the BPO-tertiary
amine system. Even polymerization at room temperature
did not give rise to maximum temperatures above 40C
(Figure 4).
Co - catalyst XC L
_____t)
DIBENZOYL PEROXIDE
BARBITURIC ACI D
derivative (R)
OXYGEN
(R)BARBITURIC ACID
OXYRADI CAL
CHLORINE- RADICAL
HYDROXYL-ANION
Figure 2. Autoxidation of (R) barbituric acid. Structures marked with
e arc able to
hyde, thus avoiding methanol present in commercial solutions to stabilize formaldehyde (59). The avoidance of
methanol as contamination benefits the preservation of fine
cellular structures.
Buffered glutaraldehyde as a single immersion fixative is
useful only when small pieces (1-2 mm3) have to be processed. In larger specimens glutaraldehyde causes a tightly
crossli~lkedproteinaceous matrix that is difficult to penetrate. As a result, the center is poorly fixed; it appears to
have been fixed only by means of a coagulating primary
fixative. This is due to the dehydration medium (eg, ethanol
or acetone). This phenomenon does not occur when tissues
have been perfi~sedwith buffered glutaraldehyde solutions.
In our laboratories we have good experiences with combinations of formaldehyde and glutar~ldehydein phosphate
buffer. In mixtures with a constant concentration formaldehyde (4%), we varied the amount of glutaraldehyde from
0.2-5%. A phosphate buffered mixture of 4% formaldehyde
and 0.2% glutaraldehyde proved to be an appropriate fixative for as well as immersion and perfusion fixation. Nerve
tissue, including ~nyelinsheaths, was especially well preserved.
The Journal of Histotechnology 1 Vol. 19, No. 4 1 December 1996
Unsuitable Fixatives
Helly and Zenker fluids are less appropriate as fixatives
for GMA embedding procedures. These fixative mixtures
contain chromiu~nions that interfere with the polymerization reaction, resulting in incomplete polymerization, especially in the center of the tissue block. Prolonged rinsing
with running tap water following fixation diminishes the
occurrence of this artifact. Osnlium tetroxide is less suitable
301
N.N - DIMETHYLANILINE
Dl BENZOYL PEROXIDE
'I
JI
CODURED
SIDE PRODUCTS
BENZOYLOXY RADICAL
BENZOIC ACID
+ CO,
PHENYL RADICAL
Peizetratioiz Problems
In many experiments we found that blood-rich tissues,
such as spleen, that were fixed in phosphate buffered formaldehyde were less easy penetrated by GMA than when
fixed with neutral aqueous formaldehyde. Similar results
were observed when tissues were fixed with the latter fixative and stored during longer periods, >14-30 days. In such
cases infiltration periods had to be prolonged to obtain satGMA Embedding for Light Microscopy I Gerrits and Horobin
90 ml
I0 ml
0.4 ml
0.5 gm
N,N-dimethylaniline
Polyethylene glycol 200
1 parts
15 parts
"Dibenzoyl peroxide moistened with 20% water or Lucidol CH-50 (dibenzoyl peroxide damped with 50% dicyclohexyl phthalate), 0.8 g/100 ml
Solution A.
isfying results. A possible explanation might be that formaldehyde in combination with a buffer effects a better
crosslinking of tissue structures, resulting in denser structures. It is well known that phosphates are effective protein
stabilizers. Besides, buffers add to the effective osmolality.
Although buffers contribute greatly to successful tissue
staining, they do not always increase tissue permeability.
Formaldehyde in its methylene glycol form penetrates tissue
quickly, but fixes slowly (62). At the same time 4% aqueous
formaldehyde possesses a high osmolality (1300 mOsm),
but the effective osmolality is low. These facts are illustrated by the observation that glycogen streaming in liver is
less evident when buffered fixatives are applied.
Minutes
Figure 5. Rat spinal cord, dorsal funiculus. Macrophages containing myclin debris in chronic experimental allergic cncephalornyelitis (Cr-EAE).
High resolution light microscopy using glycol methacrylate resin. Stain:
Sudan black B. Original magnification x1050.
Nusbickel and Swartz tested the influence of various fixatives and fixation periods upon the preservation of enzyme
activity in young embryonic tissue (67). These authors
found that fixing the tissues for 1 hr in a mixture of 95%
ethanol, 5% acetic acid and 4% neutral buffered formaldehyde resulted in retention of excellent morphology and of
alkaline and acid phosphatase. Higuchi et a1 using a combination of formaldehyde and glutaraldehyde, were not able
to demonstrate alkaline phosphatase at all in JB-4 sections,
and acid phosphatase only after prolonged incubation periods (6-24 hr) (30). This is understandable as glutaraldehyde
inhibits enzyme activity more strongly than does formaldehyde (62). The effects of fixation, processing, and acrylic
resins on the enzyme histochemical demonstration of lactase and sucrase have been evaluated by Hand (68). Formaldehyde fixation effected a marked decrease of enzyme
activity. Demonstration of activity was considerably improved following washing in gum sucrose.
At the light microscopy level formaldehyde mixtures perform well, especially in lower concentrations (46,66,69,70).
Hanschick et a1 recommend 2% formaldehyde and 5% sucrose in 0.02 M phosphate buffer (680 1n0sm) pH 7.4 as an
appropriate fixativeyor use in both enzyme histochemistry
(70). This fixative yields exceland in~~nunohistochemistry
lent preservation of enzyme activity, antigenicity and excellent morphology, even after long fixation periods (>4 hr).
Periodate-lysine-paraformaldehyde, a carbohydrate moieties stabilizing fixative, was used by Senoo in a pioneering
GMA study. This method enabled the combination of several techniques such as, routine staining, histochemistry,
immunohistochemistry, and electron microscopy on a single
biopsy block (29,7 1).
Recent studies reported the use of cold acetone (varying
from 4C to -20C) as both fixative and dehydration medium (37,72,73). Although these methods demonstrate increased levels of enzyme activity, morphological details are
impaired. Freeze-drying or freeze substitution in combination with low temperature embedding as reported by Murray
is preferable for quality of morphological detail (14). However, the use of these techniques in a routine setting has to
be questioned.
Dehydration
For routine processing schedules, increasing concentrations of ethanol are preferable. Following fixation with alm
indehydes, the type of dehydration m e d i ~ ~significantly
fluences the preservation of e n z y m e activity and
antigenicity (74). Increasing concentrations of cold acetone
(4C) or abrupt dehydration with pure cold acetone preserves several tissue antigens. At the light microscopy level,
acetone seems to be somewhat superior to ethanol as dehydration medium, preserving antigenicity better. Comparable
results can be obtained when using plasticizers such as 2isopropoxyethanol, 2-(2-methoxyethoxy)ethanol and 2-(2ethoxyethoxy)ethanol as dehydration media (46).
GMA as Dehydration Medizcrn
GMA is completely water-miscible. Dehydration with increasing concentrations of GMA leads to a better preservation of enzyme activity than dehydration with graded series
of ethanol or acetone (74). Though GMA-monomer preserves antigenicity to a greater extent morphologic detail is
not optimal, especially at the periphery of tissue blocks.
Ethanol and acetone are superior over GMA for the preservation of histologic detail. Dehydration of GMA causes
considerable tissue shrinkage and follows much the same
pattern as dehydration in ethanol, both at room temperature
and in the cold (32). This tissue shrinkage during processing
can be diminished by using GMA at low temperatures.
Cold Acetone as Well as Fixative and
Dehydration Mediurn
At this time methods utilizing cold acetone (-15C) as
primary fixative and dehydration medium are as yet to be
used roi~tinelyin in~mnocytochemistryalthough these methods excellently preserve antigenicity (14,73,75).
Microtomy
GMA-embedded tissues can be cut with different types of
knives: triangular glass knives, Ralph glass knives, disposable metal knives (Kulzer type) and knives with a D-profile
provided with a tungsten carbide cutting edge (Jung) (1 1).
The triangular glass knife offers the best c ~ ~ t t i nedge
g for
GMA embedded tissues, but this edge can be too small,
except for tissues contained in small molds (Sorvall type 12
x 6 x 5 mm). Because of the elastic properties of watermiscible resins it is advisable to use "motor drive" microtomes equipped with a retraction device, variable cutting
speed, and a variable cutting stroke. Retraction of the block
during the back stroke is crucial, thus avoiding damage and
scratches on the block surface by the knife. When using
microtomes without a retraction device, these artifacts can
easily by demonstrated after staining and mounting.
Stretching on Water, and Mounting; Some Influences
on the Final Dimensions of GMA Sections
The use of quantitative methods in microscopy requires a
knowledge of the dimensional changes that tissues and cells
undergo during histoprocessing. Such changes have been
repeatedly reviewed (15,76-79). The physical processes
that underlie resin embedding with water-miscible methacrylates are not yet fully known. The changes that take place
in liver tissue during GMA embedding are in fact minor
compared with those occurring during fixation and dehy-
dration. D ~ ~ r i ninfiltration
g
in GMA monomer there is slight
swelling (linear 2-5%), while some shrinkage occurs during
polymerization (1-2%). However, important dimensional
changes also occur during stretching of the sections on water and when mounting on slides. In both steps the physical
properties of the resin determine the ultimate dimensional
changes. It is therefore intriguing that, while a large number
of methods to mount stretched GMA sections onto glass
slides has been described, none of these authors report the
dimensional changes occurring during the procedures (91 1,29,80).
On general physical grounds, we may expect stretching of
resin sections on a water bath to be determined by the excess water-air surface tension over the sum of water-resin
and air-resin surface tensions and by the elastic properties of
the resin. When the section size reaches equilibrium, the net
s ~ ~ r f a ctension
e
is balanced by the tension in the resin section that is elastically stretched. The temperature dependence of surface tension predicts smaller size increases at
higher temperatures. The water-air surface tension can be
greatly reduced when small quantities of surface-active substances are present in the water. When one droplet of a
detergent solution is added to the water bath, a resin section
cannot be stretched in the usual way: stretching is not optimal and the section easily sinks to the bottom of the bath.
Thus, standardization of histotechnique requires constant
water bath quality. We have used distilled water. The mags
nitude of the surface tension of aqueous s o l ~ ~ t i o ncontaining solutes that do not preferentially collect at the water-air
interface, like salts and sucrose, are close to that of water.
This explains why, when using a water bath containing tap
water for stretching, virtually the same results are obtained
as with distilled water. If however even small amounts of
fatty acids or lipids are present, the surface tension decreases enormously. The corresponding molecules of such
surfactants have hydrophobic groups at one end and hydrophilic groups at the other. Cleanliness in handling the trough
and the waterbath is required (16).
Varying concentrations of a plasticizer also influenced
section stretching. Harder resin mixtures, containing less
plasticizer, stretched 13-15% (linear) at 20C and 10-12%
at 60C. Softer resin mixtures stretched about 10-13% at
20C and 7-9% at 60C. The presence of tissue slightly
influences stretching. Generally it can be stated that the
stretching values of sections of resin-embedded soft tissues
follow those of pure resin, especially at room temperature
(12,16). These results also show that the temperature
strongly influences the eventual results and that the harder
resin mixtures stretch about 3% more than the softer ones.
Correction factors have to be used in morphometrical and
stereological investigations (81). However, in the application of water-miscible resins as embedding media, it is essential to routinely use standardized procedures, since data
from dimensional changes may differ for each kind of tissue
~ , dimensional changes
and each type of cell. ~ a r t i c u l a r lthe
during stretching on water and mounting have not received
the attention they need, although these changes are of the
same magnitude as those arising during dehydration.
Micro waves
The introduction of microwave irradiation into histotechnique reduced time-consuming staining procedures by a factor of at least 10 (84). This technique is very successfi~lin
the periodic acid methenamine silver method and other
metal techniques. Because of reduced staining times, background staining of the resin matrix also decreases. However,
it has to be emphasized that microwave accelerated staining
of GMA sections can result in characteristic artifacts or
reversed staining patterns (85).
Zinpurities in GMA Sarnples
Impurities in the embedding mixture can remarkably influence the final results. It was found that excess methacrylic acid co-polymerizes with GMA, prod~~cing
a polymer
The Journal of Histotechnology / Vol. 19, No. 4 I December 1996
I
tissue-free GMA section
GMA-embedded tissue
glass slide
A'
B'
C'
Figure 6 . The influence of reagent size on the staining of GMA sections. Large dyes stain only resin-free structures exposed on the surface of uncoated
sections. Note that medium sized dyes are not readily removed from the resin matrix by aqueous washing solutions, causing marked background staining.
306
Acknowledgments
We are grateful to Audrey Glauert for her helpful and
critical comments on v a r i o ~ ~parts
s of this article. We thank
Mr. P. van der Sijde for technical assistance.
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