Journal of Histotechnology

ISSN: 0147-8885 (Print) 2046-0236 (Online) Journal homepage: http://www.tandfonline.com/loi/yhis20

Glycol Methacrylate Embedding for Light

Microscopy: Basic Principles and Trouble-Shooting
Peter O. Gerrits & Richard W. Horobin
To cite this article: Peter O. Gerrits & Richard W. Horobin (1996) Glycol Methacrylate
Embedding for Light Microscopy: Basic Principles and Trouble-Shooting, Journal of
Histotechnology, 19:4, 297-311, DOI: 10.1179/his.1996.19.4.297
To link to this article: http://dx.doi.org/10.1179/his.1996.19.4.297

Published online: 18 Jul 2013.

Submit your article to this journal

Article views: 19

View related articles

Citing articles: 5 View citing articles

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=yhis20
Download by: [Monash University Library]

Date: 15 April 2016, At: 18:24

Downloaded by [Monash University Library] at 18:24 15 April 2016

Glycol Methacrylate Embedding for Light

Microscopy: Basic Principles
and Trouble-Shooting
Peter 0. Gerrits* and Richard W. ~ o r o b i n '
:'; Department of Anatomy and Embryology, University of Groningen, The Netherlands
Department of Biomedical Science, The University, Sheffield, UK

'

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

were used for this purpose. With the introduction of epoxy

resins these methacrylates were replaced because they are
sensitive to the electron beam (3). The epoxy resins proved
more stable in this respect and also gave better morphological preservation.
Only the water-miscible resin 2-hydroxyethyl methacrylate (glycol methacrylate) remained in use, because its hydrophilic properties enabled enzyme histochemical procedures to be carried out (4). In spite of extensive applications
in electron microscopy, for many years this methacrylate
found only limited use as an embedding medium in light
microscopy. However, since the pioneering papers of Ashley and Feder, Ruddell, and Feder and O'Brien, 2-hydroxyethyl methacrylate has found its way worldwide in histotechnical laboratories (5-8).
In the literature the synonyms of 2-hydroxyethyl methacrylate (glycol methyacrylate, GMA, HEMA) have been
used interchangeably. In this review, we use the name glycol methacrylate (GMA).

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

Advantages and Disadvantages of Glycol

Methacrylate Embedding
Advarztages
The use of glycol methacrylate as an embedding medium
in light microscopy provides a large number of advantages,
when compared to embedding media such as paraffin, celloidin, and epoxy resins:
When observing the fine details of tissue structures GMA
proves to be much better than paraffin and celloidin sections
(6-14). This is partly due to GMA sections being thinner,
with consequent improvement of image resolution. In addition, GMA does not have to be removed from sections prior
to staining, preventing the distortion of tissue elements so
evident after deparaffination.

* Address reprint requests to Dr. Peter 0. Gerrits, Department of

Anatomy and Embryology, Oostersingel 69, 9713 EZ Groningen, The
Netherlands. E-mail: p.o.gerritsOmed.rug.nl

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-

The Journal of Histotechnology I Vol. 19, No. 4 1 December 1996

297

Downloaded by [Monash University Library] at 18:24 15 April 2016

tions (17). Dimensional changes during stretching and

mounting are highly reproducible (12,13,16). Resins
are therefore more suitable for quantitative investigations.
Heterogeneous tissues, including undecalcified bone,
can be sectioned at constant thickness ranging from
0.5-2.0 p m with negligible artifacts (18,19). Note,
however, that these studies dealt with GMA-methyl
methacrylate mixtures.
Staining sections of GMA is possible without removing the resin, and many histochemical staining procedures are applicable after some modification in dye
concentration or staining time. As the GMA polymer is
only very slightly hydrophobic, hydrophilic reagents
as used in most of the histochemical staining procedures are applicable (20-22). This is an obvious advantage over tissues embedded in hydrophobic media
such as epoxy resins, which are poorly accessible to
hydrophilic staining reagents. Indeed, only the partial
removal of the hydrophobic resins by dissolution or
etching can produce acceptable results with epoxy resins, as reviewed by Litwin (23). These latter procedures tend to distort the specimen morphology.
GMA monomer is considered nonreactive with ionogenic groups of tissue (20,24). However, epoxy resins
bind to tissue elements during infiltration and polymerization, with consequent modifications in tissue
chemistry (25-28). Paraffin embedding makes use of
melted paraffin (60C) during the infiltration phase,
again modifying tissue components, especially proteins; GMA embedding can be carried out in the cold
or at ambient temperature (29-31). It is probably
largely the lack of such modifications that allows enzyme activity to be demonstrated so well in GMA
sections.

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.

Applications of Resin Embedding

Resin embedding has already been recommended for routine bone marrow pathology, renal pathology, immunohistochemistry and routine pathology (18,19,3643). The applications of more specific histochemical, enzyme
histochemical and i~nmunohistochemicalmethods to resin

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.

Procedures for Embedding Tissues in GMA

(see Appendix)
Chemical and Physical Properties of Components Used
in GMA Mixtures
Principles
Embedding tissue in polymerizing resins makes use of
the principle that small liquid rnonomer molecules can peiletrate between tissue elements. Thereafter polymerization,
involving conversion of the Inonorner into long chains, can
be initiated. This can be carried out by heat; ultra violet light
in the presence of suitable free radical producing catalysts
(initiators); or by free radical producing agents, which can
be promoted by accelerators. Radicals react with the carboncarbon double bonds of the GMA molecules, forming covalent bonds. Electrons become rearranged at a different
position of the GMA molecule and a new reactive structure
is formed, which reacts with another monomer molecule,
forming a dimeric radical. Successively more monomer
molecules are added, and polymer chain radicals are
formed, and thus propagation (chain lengthening) of GMA
takes place. Propagation is terminated on reaction of pairs of
polymer radicals leading to "dead" unreactive polymer
chains. During polymerization, tissue elements become enveloped by long polymer chains. Following polymerization,
the tissues are consequently embedded within a block of
polymer. From this block 0 . 5 4 . 0 pni thick sections containing tissue may be cut with a microtome.
GMA embedding media have to be composed of several
basic components, namely: GMA-monomer, plasticizer,
and an initiator. The reactive components in an infiltration
medium are the monomer in combination with a free-radical
producing source (initiator).
GMA-Monomer
GMA in its monomeric form is the ethyleneglycol monoester of methacrylic acid. Its structural forrnula is shown in
Figure 1. The remaining hydroxyl group of ethylene glycol
is largely responsible for its hydrophilic properties. The
Hansch n value may be used as a measure of the hydrophiliclhydrophobic character of molecule fragments (44).
The Hansch n value of GMA polymer has been calculated
as weakly positive, at 0.75, so the polymer may considered
to be slightly hydrophobic. GMA lnonomer is a hygroscopic, transparent and colorless liquid with a ~nolecular
weight of 130.14; density (d:)'
1.07 1; refractive index (niO)
1.453; vapor pressure 1.3 ~ n b a (68C);
r
heat of polymerization 50 kJ/mol; flash point 101C; boiling point 250C
(1013 mbar) approx.; freezing point -60C; viscosity 5 mPa
s (30C); glass transition temperature (Tg) 55OC.
Toxicitj1 Data
GMA is a mild skin irritant and may cause allergic skin
reactions. Avoid contact with skin, eyes and do not inhale
the vapors. LD,,, 5600 mglkg (rat, oral); LD,,, 5888 mglkg

GMA Embedding for Light Microscopy / Gerrits and Horobin

'42 H2
HO-C - C -OH

ETHYLENE GLYCOL

,CH3

P
,

H2C=C

METHACRYLIC ACID

'c
\ OH

Downloaded by [Monash University Library] at 18:24 15 April 2016

/ CH3
H2C = C\ (0
C

'0-

GLYCOL METHACRYLATE
H2

C-C

H2
-OH

Figure 1. Diagram showing structures of components of GMA.

(mouse, oral). It is advisable to wear gloves when handling

GMA monomer.

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

in such a way that the amount of plasticizer lowers T g

towards room temperature.
Preparing mixtures of GMA and various plasticizers involves the miscibility of two or more substances. A quantitative approach is the calculation of the solubility parameter (6) (46). The more miscible two compounds are, the
more corresponding will be the 6 values of their solubility
parameter. The 6 values for organic liquids can be obtained
by measurement of their heat of vaporization (47). However, polymers cannot be volatilized: and determination of
their 6 value is more problematical. It can be obtained by
various calculation methods.. One method is based on the
assumption that "atomic and group increment exist which
could be summed over the known structure of the substance
(liquids as well as high molecular weight polymers), to
provide estimates for 6" (47). Moreover, when the 6 values
of a polymer and a plasticizer become more similar, the
effect of plasticization, measured by the reduction of the Tg
approaches a maximum (48).
A widely used plasticizer for experimental GMAmixtures as well as for commercial embedding mixtures (eg,
JB-4, Bio-Rad GMA kit 11, Bio-Rad Plastic Embedding media, E F L - 6 7 , and R W L ) is 2-butoxyethanol (BE)
(7,10,49,50). However, BE is a toxic irritant with a high
vapor pressure and unpleasant smell. Small molecules having a high vapor pressure are easily able to migrate from the
inside of a resin block to the surface. This results in a
noxious, unpleasant odor during sectioning, implying that
bench workers, are continuously exposed to low concentrations of a toxic irritant.
Less toxic embedding systems are now commercially
available, such as Technovit 7100, HistoResin and Technovit 8100. Technovit 7100 and HistoResin make use of barbituric acid derivatives in combination with chloride ions as
initiatorlaccelerator system and polyethylene glycol 400 as
plasticizer (Table 1) (51). Technovit 8100 makes use of
dibenzoyl peroxide as initiator (Table 2). The accelerator
solution, however, is composed of a mixture of polyethylene glycol 200 and a low concentration of a low toxicity
tertiary amine (N,N,3,5-tetramethylaniline). 2-Isopropoxyethanol is the non-toxic plasticizer (13).

Table 1. Composition of the Infiltration and

Embedding Solutions in One Commercial GMA Kit
HistoResinITechnovit 7 100 (contains glycol
methacrylate monomer, polyethylene glycol
400 as plasticizer and co-catalyst XCI)
Hardener 1 (1 bag) (contains 50% dibenzoyl
peroxide, damped with 50% dicyclohexyl
phthalate)

100 ml

1 gm

Enzbeddiizg Solution
Infiltration Solution A
Hardener I1 (contains the accelerator solution,
a barbituric acid derivative)

15 ml
1 ml

Table 2. Low Toxicity Embedding Medium RES

G20/(Technovit 8100)
Solution A (RES G20
I~zJiltratioizSolutioiz)
Glycol methacrylate
(200-300 ppm
hydroquinone monomethyl
ether)
2-Isopropoxy ethanol
Ethylene glycol
dimethacrylate
Lucid01 CH-50 (dibenzoyl
peroxide damped with
50% dicyclohexyl
phthalate)

90 ml

(monomer)

10 ml
0.4 ml

(plasticizer)
(crosslinker)

0.6 gm

(initiator)

Downloaded by [Monash University Library] at 18:24 15 April 2016

Solutioiz B (RES G20

Accelerator Solutioiz)
N,N,3,5-tetramethylaniline
Polyethylene glycol 200
(carrier for the
accelerators)

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-

tiary aromatic amines has been explored by several groups

(5455). N,N-dimethylaniline is now widely used in commercial GMA embedding kits and in self-prepared GMAmixtures (Table 3). However, DA has serious toxic properties. In RES G20 (Table 2) and Technovit 8 100 this tertiary
amine was replaced by N,N,3,5-tetramethylaniline, which
may be considered less toxic (13).

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).

Fixation of Tissues for Resin Embedding

Fixation in Routine Practice
As fixing agents aldehydes (eg, formaldehyde and glutardialdehyde in buffered and unbuffered solutions) are preferable. In routine practice we favor a 0.1 M phosphate buffered solution of 4% formaldehyde pH 7.4. When enzyme
histochemistry has to be carried out, cacodylate may be the
buffer of choice. This buffer prevents precipitation of salt
complexes in metal salt procedures for phosphatases (eg,
acid phosphatase, ATP-ase, 5'-nucleotidase) (57,58).
Formaldehyde has to be prepared from paraformaldeGMA Embedding for Light Microscopy I Gerrits and Horobin

Co - catalyst XC L
_____t)

DIBENZOYL PEROXIDE
BARBITURIC ACI D
derivative (R)

Downloaded by [Monash University Library] at 18:24 15 April 2016

OXYGEN

(R) BARBITURIC ACID

HYDROPEROXIDE

(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

initiate the polymerization of glycol methacrylate

To demonstrate glycogen in liver tissue by means of the

PAS-reaction, it is advisable to use phosphate buffered
formaldehyde. Using unbuffered formaldehyde as primary
fixative results in streaming artifacts. This artifact can be
avoided for the major part when cold (4C) buffered formaldehyde is used. The combination of buffered formaldehyde and glutaraldehyde guarantees excellent morphologic
detail: well preserved nuclei and a sharply defined cell wall.
Bear in mind, however, that when only one end of the
glutaraldehyde molecule is involved in fixation, the other
aldehyde group is free to react with Schiff reagent, thus
inducing false positive staining results with the PAS or the
Feulgen reaction.

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

Downloaded by [Monash University Library] at 18:24 15 April 2016

N.N - DIMETHYLANILINE

Dl BENZOYL PEROXIDE

'I

JI

CODURED
SIDE PRODUCTS
BENZOYLOXY RADICAL

BENZOIC ACID

+ CO,
PHENYL RADICAL

SUPPRESSION OF THE ACTIVITY OF

N.N-DIMETHYLANILI NE
Figure 3. Deco~npositionof dibenzoyl peroxide in the presence of N,N-dimethylaniline.

as a fixative for tissue embedding in GMA. Being partly

linked to the tissue, osmium tetroxide reacts with the double
bonds of GMA-monomer, thus leading to inhibition of the
polymerization inside the tissue block. Recently, it was reported that the combination of osmium tetroxide and GMA
is still possible in very thin tissue pieces or vibratome sections (61,62). The resulting sections show excellent morphological detail and high color contrasts (Figure 5).

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

Table 3. Composition of the Recommended RES GI0

Infiltration Solution (A) and Accelerator Solution (B)
Sol~itioilA
Glycol methacrylate
2-Butoxyethanol
Ethylene glycol dimethacrylate
Dibenzoyl peroxide':'

90 ml
I0 ml
0.4 ml
0.5 gm

N,N-dimethylaniline
Polyethylene glycol 200

1 parts
15 parts

Downloaded by [Monash University Library] at 18:24 15 April 2016

"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.

Fixation in Enzyme Histochenzistry

Several methods have been developed to obtain satisfactory preservation of enzyme activity in tissues embedded in
GMA (eg, freeze-drying or freeze-substitution) (63-66).
Freeze-drying in combination with low-temperature embedding permits the visualization of a wide range of enzymes
(oxidoreductases, esterases, peptidases, phosphatases and
dehydrogenases) with precise enzyme localization, high enzyme activity, and excellent tissue morphology (14,66).

Minutes

Figure 4. A comparison of thermal kinetics for the two common types of

GMA polymerization systems. The solid curve shows thermal effects during polymerization, using dibenzoyl peroxide and N,N-dimethylaniline as
initiator-accelerator system. A marks the moment paraffin was poured
around the blockholders. The dotted line shows the same effect for Technovit 7100/HistoResin. This latter system makes use of a barbituric acid
derivative in combination with chloride ions as initiator-accelerator system.

The Journal of Histotechnology / Vol. 19, No. 4 1 December 1996

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.

Downloaded by [Monash University Library] at 18:24 15 April 2016

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.

Staining of GMA-Embedded Tissue Sections

We can be state that routine and histochemical staining
proced~~res
applicable to paraffin and frozen sections can be
GMA Embedding for Light Microscopy IGerrits and Horobin

Downloaded by [Monash University Library] at 18:24 15 April 2016

carried out with minor modifications. Sometimes only the

staining time has to be increased or the staining temperature
to be elevated. Incidentally the concentration of the dye has
to be increased (eg, trichrome stains; Gill hematoxylin,
triple strength). One category of stains, in particular the
trichro~nestains for connective tissue, appeared inadequate.
Trichrome staining solutions containing mordants like phosphomolybdic acid or phosphotungstic acid cause the GMA
sections to wrinkle and detach the sections from the glass
slide. In our hands the modified trichrome staining procedure according to Osheroff and Ruffing proved to yield
satisfying reproducible results (82). This method implies a
pretreatment of the sections with a prewarmed Bouin solution. An acid hydrolysis with picric acid is probably preferable over the above mentioned mordants. Blaauw et a1
reported a simple, but reliable, rapid connective tissue stain
for GMA embedded tissue (83). The application of Schiff
reagent in histoche~nicalprocedures like Feulgen and PAS
is rather unproblematic. However, PAS following diastase
will demonstrate an aspecific staining pattern. Because of its
size (>I500 Da) the enzyme will exert its action only at the
su~faceof the GMA section. Deeper lying PAS-positive
tissue structures remain unaffected. Schiff reagent (ionic
weight 370 Da) will specifically stain such structures.

Peizetration of Dyes into Sectioizs

Dye size is a major factor in controlling penetration of
resins and staining of tissues. Large dyes (>I000 Da) enter
GMA very slowly, and stain only those tissue components
poorly infiltrated by resin. However, small dyes (<550 Da)
penetrate GMA readily and stain tissue components whether
or not they were resin-infiltrated. Dyes of intermediate size
(550 < MW < 1000 Da) penetrate the resin, but the staining
of resin-infiltrated tissue elements is slow.
Background staining of resin also varies with dye size.
Large dyes do not stain tissue-free GMA. Small dyes do, but
are readily removed by water. Dyes of intermediate size
penetrate resin slowly, and once inside are lost slowly. This
produces background staining, which requires the use of
plasticizing solvents such as ethanol for its removal. Increases in crosslinks also reduce staining rates. All these
observations are consistent with diffusion rate being controlled by GMA. For a diagrammic summary of these conclusions see Figure 6. Detailed information about this rate
control model of staininglde-staining has been published
previously (2 1-22).

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

matrix with fi-ee carboxylic groups. These groups, when

dissociated (pH >4), react with cationic dyes such as toluidine blue and ~nethyleneblue, producing undesirable background staining of the resin matrix. The presence of large
amounts of crosslinkers (eg, ethyleneglycol dimethacrylate)
may lead to reduced rates of penetration of dyes into GMA,
and, as a result, in reduced staining (21). Highly pure GMAmonomers are used for preparing Technovit 7100,
HistoResin and Technovit 8100. These mixtures, when
stored for more than 2 years, do not decompose into undesirable impurities (45).

Zizstability of GMA Sectioizs

GMA sections are unstable in alcoholic and basic staining
solutions, both tending to loosen the sections from the glass
slide. An example of a staining procedure that may cause
difficulties is the periodic acid methenamine silver method.
During prolonged staining periods (>1 hr) in the heated
silver solution, the sections easily dislodge from the slide.
This can be avoided by coating the glass slides with Mayer
albumen prior to mounting and drying the sections or by
using microwaves. The Alcian blue method according to
Archimbaud et a1 can also be successf~~lly
used for attaching
GMA sections to glass slides (86).
Following hematoxylin-eosin staining of GMA embedded tissue sections, sections often exhibit "mini-folds."
These mini-folds are very characteristic (Figure 7) and
highly reproducible (50). Initially these mini-folds were attributed to use of a combination of hematoxylin a ~ l dalcoholic eosin. However, they were not seen with every sample
of GMA, but only in the purest ones. Thus, certain impurities in the various GMA samples are responsible for an
absence of these mini-folds. Only sections prepared from
samples of GMA containing no or minimal concentrations
of ethyleneglycol dimethacrylate (EDMA, a crosslinker),
showed these mini-folds. However, when samples of GMA
containing increased amounts of EDMA were used, sections
did not show these mini-folds. It is evident that rninimal
amo~lntsof a crosslinker improve the stability of resin sections in staining solutions. Sections prepared from Technovit 7100, HistoResin, RES G10, RES G20, and Technovit
8 100 do not show mini-folds after hematoxylin-eosin stainIng.
Use of Tissue-Free GMA Sections as
Serni-Perineable Membraltes
Placing 2 p,m sections of tissue-free GMA on top of
mounted tissue sections is a simple way to use semipermeable membranes in enzyme histochemistry (87). As to
the demonstration of alkaline phosphatase by standard azo
dye methods, GMA membranes enable inc~tbationtimes to
be reduced to 1-2 hr. The azo dye reaction product is more
crisply localized as compared to sections without GMA
membranes. Moreover, diffusion, fading and crystalization
of the final reaction product are prevented for the most part.
Such effects are possible because GMA membranes are
highly permeable to small substrate molecules, slightly permeable to the moderate sized reaction product, and impermeable to large molecules such as alkaline phosphatase and
bio-polymers.
Use of GMA Resins in Iinmuizohistochenzistry
Adaptations may be required for immunohistochemical
procedures to be carried out successfully with GMA sec-

I
tissue-free GMA section
GMA-embedded tissue
glass slide

Downloaded by [Monash University Library] at 18:24 15 April 2016

Small dyes, ionic weight < 550 Da

Intermediate-sized dyes, 550 < ionic weight < 1000 Da

Large dyes, ionic weight > 1000 Da

A'

B'

C'

- GMA-embedded tissue stained with single dyes ( 1 rnin ).

- Comparable sections but coated with a superficial layer
of tissue-free GMA.

A" B" C"

Sections following a poststaining water rinse ( 1 min ).

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

GMA Embedding for Light Microscopy 1 Gerrits and Horobin

Downloaded by [Monash University Library] at 18:24 15 April 2016

Figure 7. Mini-folds resulting from instability of GMA sections are

clearly detectable by simple staining methods. 2 pi11 section of rat intestine,
stained with Gill hematoxylin and eosin. Original inagnification x330.

tions various. These may involve just the embedding step or

other histoprocessing stages (88). Thus fixation fluids may
be modified. Examples of this include lowering formaldehyde concentration and shortening fixation time. Processing
may also be altered: processing periods may be reduced and
processing may be carried out in the cold. With specific
regard to immunohistochemistry, it is advisable to use cold
acetone absolute instead of ethanol as dehydration medium.
To diminish loss of enzyme activity or antigenicity due to
exothermic heat, polymerization may be carried out on
crushed ice. Peak temperatures above 40C are harmful to
antigenic sites.
Technovit 8100, making use of BPO in combination with
a less toxic tertiary amine as initiatorlaccelerator system,
was found to give more consistent results than Technovit
7 100 and HistoResin (13). When using the Technovit 8 100
mixture we consistently demonstrated high levels of enzyme activity, as well as a broad spectrum of antigens
(13,89,90).
An acceptable explanation for the difficulties and their
possible elimination by immunohistochemical staining of
GMA embedded tissue can be found by combining the work
of Horobin and Wright (20,24). Horobin disc~~ssed
staining
of resin sections and reviewed the problems that could occur. The major part of this work concerns hydrophobic resins (Araldite and epoxides), mainly used in EM. However,
some underlying similarities of hydrophobic and hydrophilic resin embedding media are dealt with a section in
which he made generalizations: The constitution of tissue
structures is heterogeneous (physically dense, loose, or
highly hydrated). In addition, tissue structures can be hydrophilic or hydrophobic. It is understandable that all these
variations in tissue properties lead to heterogeneous penetration of resins into tissue during the infiltration phase
prior to polymerization. In the application of a controlled
enzymatic digestion, the enhancement of immunolabeling
can be explained by the fact that poorly embedded tissue
structures are affected, resulting in surface enlargement. As
a consequence, a greater efficiency in formation of antigenantibody complexes {nay result. Another, surely no less important, statement is that protein crosslinks caused by the
fixative are cleaved by controlled digestion with proteases,
The Journal of Histotechnology I Vol. 19, No. 4 1 December 1996

making more antigenic sites available to the antibody

(9 1,92). Incubation with proteolytic enzymes should be varied in accordance with the fixative mixt~lreused and the
antigen to be determined. A too-long proteolytic digestion
understandably decreases antigenicity (93). Undoubtedly
digestive actions have additive effects in immunostaining.
Up to now, there has not been any water-miscible resin
mixture that can completely be removed from the tissues
after polymerization. If there were, fewer problems would
be expected at the light microscopy level.
In conclusion, it has to be emphasized that extensive
understanding of the fundamental aspects of water-miscible
methyacrylates in light microscopy is necessary when histochemical-, enzyme histochemical- and immunohistochemical techniques are to be carried out. It would, therefore, b e desirable that firms producing commercial
embedding mixtures disclose the composition of their products to a certain extent. Only a thorough knowledge of their
properties will guarantee that water-miscible methacrylates
acquire a permanent key position in biological and clinical
research.

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.
References
1. Klebs: Die Einschmelzungs-Methode ein Beitrag zur mikroskopischen Technik. Arch f Mikrosk Aizat 5: 164-166, 1869
2. Duval M: Technique de I'emploi du collodion humide pur la
pratique des coupes microscopiques. Jo~~rnal
rle 1 'nnnt et de In
plzysiol T XV p 185, 1879
3. Glauert AM, Rogers GE, Glauert RM: A new embedding
medium for electron microscopy. Nature (Lorlcloiz) 178:803,
1956
4. Leduc EH, Bernhard W: Water-soluble embedding media for
ultrastructural cytochemistry. Symp Iizt Soc Cell Biol 121-28,
1962
5. Ashley CA, Feder N: Glycol methacrylate in histopathology.
Arch Pnthol 81:39 1-397, 1966
6. Ruddell CL: Hydroxyethyl methacrylate combined wtih polyethylene glycol 400 and water; an embedding medium for
routine 1-2 micron sectioning. Stnirz Teclzrlol 42:l 19-123,
1967a
7. Ruddell CL: Embedding media for 1-2 micron sectioning 2
Hydroxyethyl methacrylate combined with 2-butoxyethanol.
Stain Techno1 42253-255, 1967b
8. Feder N, O'Brien TP: Plant microtechnique: some principles
and new methods. Am J Bot 55(1):123-142, 1968
9. Cole MB Jr, Sykes SM: Glycol methacrylate in light microscopy: a routine method for embedding and sectioning animal
tissues. Stnirz Techno1 49:387-400, 1974
10. Sims B: A simple method of preparing 1-2 micron sections of
large tissue blocks using glycol methacrylate. J Microsc 101:
223-227, 1974
I I. Bennett HS, Wyrick AD, Lee SW, McNeil JH Jr: Science and
art in preparing tissues embedded in plastic for light microscopy with special reference to glycol methacrylate glass
knives and simple stains. Stain Techno1 5 1 :7 1-97, 1976
12. Hanstede JG, Gerrits PO: The effects of embedding in watersoluble plastics on the final dimensions of liver sections. J
Microsc 131 :79-86, 1983
13. Gerrits PO, Eppinger B, Van Goor H, Horobin RW: A versatile low toxicity glycol methacrylate embedding medium for

Downloaded by [Monash University Library] at 18:24 15 April 2016

use in biological research and for recovered bio~naterialsprostheses. Cells Mcrter-icrls l(3): 189- 198, 1991
14. Murray GI: Enzyme histochemistry and irl~munohistochetnistry with freeze-dried or freeze-substituted resin-embedded tissue. Hi.stoc1zern J 24:399408, 1992
15. Iwadare T, Mori H, Ishiguro K, Takeishi M: Di~nensional
changes of tissues in the course of processing. J Microsc
136:323-327, 1984
16. Gerrits PO, Van Leeuwen MBM, Boon ME, Kok LP: Floating
on a water bath and mounting glycol methacrylate and hydroxypropyl methacrylate sections influence final ditnensions.
J Microsc 145: 107-1 13, 1987b
17. Helander KG: Thickness variations within individual paraffin
and glycol methacrylate sections. J Microsc 132223-227,
1983
IS. Westen H, Muck KF, Post L: Enzyme histochemistry on bone
marrow sectiotls after embedding in methacrylate at low temperature. Histoclzenzi.stry 70:95-105, 198 1
19. Chappard D, Alexandre C, Catnps M, Montheard JP, Riffat G:
Embedding iliac bone biopsies at low temperature using glycol and methyl methacrylates. Stnir~ Techrlol 58:299-308,
1983
20. Wright DJ: blvestigcrtir~~
tlze rrrtior~nluse of JP-4 ernberlrlirlirlg
inerli~rtil,for irse in light rilicroscopy. Thesis submitted in partial fulfilltnent of requirements for a BSc Sheffield University
Dept of Anatomy and Cell Biology, 1985
21. Gerrits PO, Horobin RW, Wright WJ: Staining sections of
water-miscible resins. 1. Effects of the molecular size of the
stain and of resin cross-linking on the staining of glycol methacrylate embedded tissues. J Microsc 160279-290, 1990b
22. Horobi~lRW, Gerrits PO, Wright DJ: Staining sections of
water-miscible resins. 2. Effects of staining-reagent lipophilicity on the staining of glycol-methacrylate-etnbedded tissues. J
Microsc 166:199-205, 1992
23. Litwin JA: Light n~icroscopichistochemistry on plastic sections. In Progress irl Hi.stocl~errzi.str:\l ai~clC~~tochernistr-y,
Vol
16, No 2. Gustav Fisher Verlag, Stuttgart, 1985
24. Horobin RW: Staining plastic sections: A review of explanations and possible solutions. J Microsc 131: 173-1 86, 1983
25. Fisch W, Hoffmann W, Koskikallio J: The curing mechanism
of epoxy resins. J AppI Cllerrl 6:429-44 1, 1956
26. Lee H, Neville K: Epoxy Resirls: The Applicntiorl nr~rlTechr~ology.McGraw-Hill, New York, 1957
27. Stacey KA, Cobb M, Cousens SF, Alexander P: The reaction
of the "radiomimetic" alkylating agents with the macromolecules in vitro. Anti NY Acncl Sci 68:682-701, 1958
28. Takamiya H, Batsford SR, Tok~tnagaJ, Vogt A: Imtnunohistological staining of antigens on setnithin sections of specimens embedded in plastic (GMA-Quetol 523). J Itnm~rr~ol
Meth 30277-288, 1979
29. Senoo A: A new preparation method for several histopathological examinations on a single block. Stcrir~Teclzr10153:123129, 1978
30. Higuchi S, Suga M, Dannenberg AM Jr, Schofield BH: Histochemical demonstration of enzyln activities in plastic and
paraffin embedded tissue sections. Stair1 Techrlol 54:5-12,
1979
3 1. Namba M, Dannenberg AM Jr, Tanaka F: Improvetnent in the
histochetnical demonstration of acid phosphatase betagalactosidase and nonspecific esterase in glycol methacrylate
tissue sections by cold temperature embedding. Srniiz Techno1
58:207-213, 1983
32. Gerrits PO, Horobin RW, Stokroos I: The effects of glycol
methacrylate as a dehydration agent on the dimensional
changes of liver sections. J Microsc 165:273-280, 1992
33. Cope GH, Willia~nsMA: Quantitative studies on neutral lipid

preservation in electron microscopy. J Roy Micr Soc 88:259277, 1968

34. Van Goor H, Gerrits PO, Grond J: The application of lipidsoluble stains in plastic-ernbedded sections. Histochernistry
85:25 1-253, 1986
35. Chappard D: Uniform poly~nerizationof large blocks in glycol
methacrylate at low temperature with special reference to enzyme histochemistry. Mikr-oskopie 42148-150, 1985
36. Beckstead JH, Halverson PS, Ries CA, Bainton DF: Enzyme
histochemistry and i~nrnunohistochemistryon biopsy specimens of pathologic human bone marrow. Bloorl6: 1088-1 098,
1981
37. Jeffree GM, Quilty RD: Phosphatase staining of semi-thin
sections of bone. Histocher~lJ 14521-522, 1982
38. Mostafa EA, Meyer RA Jr, Latorraca 0 : A simple and rapid
method for osteoclast identification using a histochemical
method for acid phosphates. Histochern J 14:409413, 1982
39. Agodoa LCY, Striker GE, Chi E: Glycol methacrylate embedding of renal biopsy specimen for light microscopy. A111J Clit~
Patlz 64:655-660, 1975
40. Beckstead JH: Optimal antigen localization in human tissues
using aldehyde-fixed plastic-embedded sections. J Hisrocllern
Cytoclzer~l9954-958, 1985
41. Van Goor H, Harms G, Gerrits PO et al: I~nrnunohistochemical antigen demonstration in plastic ernbedded lymphoid tissue. J Histocheril Cytochern 36: 1 15-120, 1987
42. Lumb G: Plastic embedding as a tool in surgical pathology
diagnosis. Arzrl Clin rrizcl Lab Scicrlce 13:393-399, 1983
43. Poppenla S: Plastic embedment in routine pathology. Verh
Dtsch Ges Path 67:20-22, 1983
44. Hansch C, Leo A, Unger A, Kim SH, Lien EG: Aromatic
substituent constants for structure-activity correlations. J Mecl
Clzer~16: 1207-1216, 1973
45. Gerrits PO, Eppinger B: Effects of storing initiated 2-hydroxyethyl methacrylate solutions for etnbcdding tissues for light
microscopy: sorne practical implicatiotls. Biotechr~icHistoclleir~70: 155- 163, 1995
46. Gerrits PO, Horobin RW, Hardonk MJ: A nu~nericalprocedttre for choosing effective low toxicity plasticizers for glycol
methacry late embedding. Histoehein J 224391.5 1, 199Oa
47. Fedors RF: A method for estimating both the solubility parameters and molar volu~nesof liquids. Polyrller Eng Sci 14:
147-1 54, 1974
48. Ingatnells WC: Important cotlcepts in the dyeing and finishing
Man-made fibres. JSDC 96:4661.74, 1980
49. Gerrits PO, Van Leeuwen MBM: Glycol methacrylate embedding in histotechnology: factors which influence the evolutiotl
of heat during polymerization at room temperature. J Microsc
139:303-3 1 1, 1985
50. Gerrits PO, Van Leeuwen MBM: Glycol methacrylate embedding in histotechnology: the hematoxylin-eosin stain as a
method for assessing the stability of glycol methacrylate sections. Strrirl Tech110162: 18 1-190, 1987a
5 1. Gerrits PO, Srnid L: A new less toxic polymerization system
for the embedding of soft tissues in glycol rnethacrylate and
subsequent preparing of serial sections. J Microsc 132:81-85,
1983
52. Bredereck H, Petranyi P, Posselt K, Sigel H: ~ b e CH-aktive
r
Polytnerisations Initiatoren .XI11 Mitt Polymerisationen und
Polymerisationsinitiatoren. Mrrkror~lolClzer~z
9270-90, 1966
53. Gross A: 1st die Verwendung von Kaltpolytnerisat bei der
Herstellung definitiver Prothesen nach detn heutigen Entwicklungsstand zu vertreten? Q~lirztessei~z
rl Zrrhr~techr~ik
7:45-50,
1977
54. Horner L, Junkermann H: Studien zutn Ablauf der Substitution VII Beitrag zum kinetischen Nachweis von Durchgangsradikalen im System: Tertiares Amin-Dibenzoyl-peroxid.
Arlrl Clzemie 59 153-68, 1955
GMA Embedding for Light Microscopy / Gerrits and Horobin

Downloaded by [Monash University Library] at 18:24 15 April 2016

55. Imoto M, Choe S: Vinyl polymerization V Decomposition of

sym-substituted benzoyl peroxides in the presence of dimethylaniline. J Polyrn Sci 15:485-501, 1955
56. Ashford EA, Allaway WG, Gubler F et al: Temperature control in Lowicryl K4M and glycol methacrylate during polymerization: is there a low-temperature embedding method? J
Microsc 144: 107-126, 1986
57. Gomori G: Microscopic Histochernistty: Principles ancl Pmctice. The University of Chicago Press Chicago, 1952
58. Wachstein M, Meisel E: Histochemistry of hepatic phosphatases at a physiologic pH. Atner J Clin Path 27:13-23, 1957
59. Karnovsky MJ: A formaldehyde-glutaraldehyde fixative of
high osmolarity for use in electron microscopy. J Cell Biol
27:137a-138a, 1965
60. Gerrits PO, Brekelmans-Bartels M, Mast L, 's-Gravenmade
EJ, Horobin RW, Holstege G: Staining myelin and myelinlike degradation products in the spinal cords of chronic experimental allergic encephalomyelitis (Cr-EAE) rats using Sudan Black B staining of glycol methacrylate-embedded
material. J Neurosc Meth 45:99-105, 1992
61. Gerrits PO, Holstege G: Staining myelin and myelin-like
products: glycol methacrylate resin-embedding combined
with Sudan black B staining. Ne~iroscProt 95-050-01-01-1 1,
1995
62. Hopwood D: Some aspects of fixation with glutaraldehyde A
biochemical and histochemical comparison of the effects of
formaldehyde and glutaraldehyde fixation on various enzymes
and glycogen with a note on penetration of glutaraldehyde into
liver. J Anat 10133-92, 1967
63. Mitrenga D, Arnold W, von Mayersbach H: Freeze-drying and
embedding in glycol methacrylate (GMA) The results of morphological and immunohistological investigations. Histoclzemistry 39:3 13-326, 1974
64. Arnold W, Mitrenga D, Von Mayersbach H: Gefriertrocknung
und Einbettung in Glykolmethacrylat (GMA): Ergebnisse histochemischer Reaktionen. Acta Histochetn Suppl Bd 14:271277, 1975
65. Hopfel-Kreiner I, Von Mayerbach H: The influence of glycol
methacrylate (GMA) and paraffin embedding on freeze substituted and fixed tissues for enzyme histochemistry. Acta Histochem 63:224-234, 1978
66. Murray GI, Burke MD, Ewen SWB: Dehydrogenase enzyme
histochemistry on freeze-dried or fixed resin-embedded tissue.
Histoclzem J 20:491-498, 1988
67. Nusbickel FR, Swartz WJ: Enzyme histochemical investigation of glycol methacrylate embedded chick embryonic tissue.
Histoclzenz J 11: 197-203, 1979
68. Hand NM: Enzyme histochemical demonstration of lactase
and sucrase activity in resin sections: the influence of fixation
and processing. Med Lab Sci 45:125-130, 1988
69. Pretlow TP, Lapinsky AS, Flowers LC et al: Enzymatic histochemistry of mouse kidney in plastic. J Histochet7z Cytocherrz 35:483487, 1987
70. Hantschick M, Wolf E, Dominok G: Einfluss der Fixation
Dehydrierung und Polymethacrylateinbettung auf die Ergebnisse immun- und enzymh~stochemischerUntersuchungen am
lymphatischen Gewebe und Knochenmark. Actu Hi.stoclzem
35: 165-177, 1988
7 1. McLean IW, Nakane PW: Periodate-lysine-paraformaldehyde
fixative; a new fixative for immunoelectron microscopy. J
Histochei~zCytochem 22: 1077-1083, 1974
72. Randall HW, Bogdanffy MS, Morgan KT: Enzyme histochemistry of the rat nasal mucosa embedded in cold glycol
methacrylate. At~zJ Anat 179: 10-17, 1987
73. Van Goor H, Gerrits PO, Hardonk MJ: Enzyme histochemical
demonstration of alkaline phosphatase activity in plasticembedded tissues using Gomori-based cerium-DAB technique. J Histoclzenz Cytoclzem 37:173-176, 1989
The Journal of Histotechnology IVol. 19, No. 4 1 December 1996

74. Gerrits PO, Zuideveld R: The influence of dehydration media

and catalyst systems upon the enzyme activity of tissues embedded in 2-hydroxyethyl methacrylate: an evaluation of three
dehydration media and two catalyst systems. Mikroskopie 40:
321-328, 1983
75. Casey TT, Cousar JB, Collins RD: A simplified plastic embedding and imrnunohistological technique for immunophenotypic analysis of hematopoietic and lymphoid tissue. Am J
Path01 131: 183-189, 1988
76. Bahr GF, Bloom G, Friberg U: Volume changes of tissues in
physiological fluids during fixation in osmium tetroxide or
formaldehyde and during subsequent treatment. Ex11 Cell Res
121342-355, 1957
77. Baker JR: Principles of Biological Microtechnique: A Study of
Fixatiorz and Dyeirtg. Methuen, London, 1960
78. Glauert AM: Fixation dehydration and embedding of biological specimens In Practical Methods in Electrotz Microscopy,
Vol 3. North Holland, Amsterdam, 1974
79. Boyde A, Maconnachie E: Treatment with lithium salts reduces ethanol dehydration shrinkage of glutaraldehyde fixed
tissue. Histoclzemistty 66: 181-187, 1980
80. Ruddell CL: Embedding media for 1-2 micron sectioning
hydroxyethyl methacrylate-benzoyl peroxide activated with
pyridine. Stain Techrzol 46:77-83, 1971
8 1. Weibel ER: Stereological Methods, Vol I : Practical Methods
for Biological Morplzometty. Academic Press, London, 1979
82. Osheroff MR, Ruffing RN: An improved trichrome procedure
for glycol methacrylate-embedded lung tissue. J Histotechr~ol
8192-94, 1985
83. Blaauw EH, Jonkman MF, Gerrits PO: A rapid connective
tissue stain for glycol methacrylate embedded tissue. Acta
Morphol Neerl- Scarzd 25:167-172, 1987
84. Boon ME, Kok LP: Microwave Cookbook of Patlzology: Tlie
Art of Microscopic Visualisation. Coulomb Press, Leyden,
1987
85. Horobin RW, Boon ME: Understanding microwavestimulated Romanowsky-Giemsa staining of plastic embedded
bone marrow. Histochenl J 20:329-334, 1988
86. Archimbaud E, Islam A, Preisler HD: Alcian blue method for
attaching glycol methacrylate bone marrow sections to glass
slides. Stain Techno1 61: 121-122, 1986
87. Gerrits PO, Horobin RW, Hardonk MJ: Use of tissue-free
glycol methacrylate sections as semi-permeable membranes: a
simple way to shorten incubation times and to improve localization in enzyme histochemistry. J Histoclzenz Cjltochetn 37:
173-1 76, 1989
88. Gerrits PO, Van-Goor H: Immunohistochemistry on glycol
methacrylate embedded tissues: Possibilities and limitations. J
Histotechrzol 11:243-246, 1988
89. Van Goor H, van der Horst LC, Fidler V, Grond J: Glomerular
macrophage modulation affects mesangial expansion in the rat
after renal ablation. Lab Invest 66564-571, 1992
90. Van Goor H, van der Horst LC, Atmosoerodjo J et al: Renal
apolipoprotein in nephrotic rats. Am J Patho1142: 1804-1 8 12,
1993
9 1. Polak JM, Van Noorden S: Itnm~tnocytnchet~zi.vtty:
Practical
Applications in Pathology and Biology. Wright, Bristol, 1983
92. Litwin JA, Yokota S, Hashimoto T, Fahimi HD: Light microscopic immunocytochemistry of peroxisomal enzymes in
epon-embedded rat liver. Histochemistty 8 1;15-22, 1984
93. Casanova S, Donini U, Zini N et al: Immunohistochemical
staining on hydroxyethyl-methacrylate embedded tissues. J
Histoclzern Cytoclzet~z3 1: 1000-1004, 1983
94. Cole MB Jr: Methods and results of testing "low acid" glycol
methacrylate (GMA) for light microscopic cytochemistry. J
Histochem Cytocher~z32:555-556, 1984

Downloaded by [Monash University Library] at 18:24 15 April 2016

Appendix: Procedures for Embedding Tissues in GMA

GMA Resin
Use a GMA product that has an inhibitor (either hydroquinone or hydroquinone monomethyl ether) concentration
in the range of 200-300 ppm. Measure the pH of the GMA
as 10% solutions of the monomer in distilled water, as described by Cole (94). Those with pH values <4 are unsuitable.
1. Prepare a tissue-free GMA block, using materials
specified in Table 2 or 3. From this cut a number of 2
pm sections.
2. Check some sections for background staining after
staining with 1% Inethylene blue in 1% borax (2 min).
Rinse with tap water (1 min) to remove excess stain.
Satisfactory resins will give sections that are glass
clear; if sections are colored, use a GMA monomer
giving a higher pH (see above).
3. To reveal the presence of mini-folds, stain some sections with hematoxylin (Gill)-eosin. If mini-folds are
detectable, add a small amount of the crosslinker ethylene glycol dimethacrylate to the infiltration solution. In most cases, 0.4-0.5 m11100 ml prove to be
sufficient. By rejecting GMA giving background
staining or showing mini-folds, a high quality GMA
sample is now available to embed tissues.

A. Processing Schedule for Benzoyl Peroxide-Tertiary

Amirze System-Initiator-Accelerator System
This initiator accelerator system is used in several commercial embedding kits (eg, JB-4; Bio-Rad GMA kit 11;
Bio-Rad Plastic Embedding media; RWL and EFL-67).
Method
1. Fix tissues, of maximum size 10 x 10 x 2 mm, in 4%
formaldehyde or 4% formaldehyde in 0.1 M phosphate buffer at pH 7.4. Prepare the formaldehyde
from paraformaldehyde (Aldrich Chemical Company, Milwaukee, WI) according to the method described by Karnovsky (59).
2. Dehydrate the tissue with increasing concentrations
of ethanol: ethanol 70% (2 hr), 2 changes of ethanol
96% (2 h each) then absolute ethanol (I hr).
3. Pre-infiltrate in equal amounts of absolute ethanol
and infiltration RES G10 Solution A (Table 3) for
2 hr.
4. Infiltrate the tissue with RES G I 0 Solution A (overnight, 18 hr). The infiltration time may be shortened
with smaller specimens.
5. Prepare the embedding Solution C by adding 1 part
of Solution B to 30 parts of Solution A, and stir for
1 min.
6. Place the tissue blocks into plastic embedding molds
(Sorvall type 12 x 16 x 5 mm or 12 x 6 x 5 mm)
containing embedding Solution C, and fit the aluminum block holders.
7. Pour melted paraffin wax (60C) around the blockholders to exclude atmospheric oxygen, which acts
as a polymerization inhibitor.
8. Polymerize at room temperature for 2-4 hr. Dispersal

of exothermic heat may be facilitated by placing the

embedding molds in cold water, in a refrigerator, or
on cracked ice.
9. Remove the blocks from molds and cut sections. Sectioning under standardized conditions is advisable;
preferably in a temperature controlled room (20C),
with 60% relative humidity.
10. Stretch sections on distilled water and mount them
on clean glass slides. Dry sections at room temperature or on a hotplate at 60C.

B. Processiitg Schedule for HistoResin and

Techrzovit 7100
The HistoResin and Technovit 7100 embedding kits
make use of the barbituric acid-choride ion polymerization
system (5 1). These kits comprise two solutions, the monomerlplasticizer mixture and a solution of a barbituric acid
derivative (Hardener II), and a damped dibenzoyl peroxide
(Hardener I).
Method
1. Process tissues as described in schedule A, steps 1-4.
2. Prepare the embedding solution specified in Table 1
and stir for 1 min.
3. Place the specimens in the embedding molds containing the embedding solution, and fit the blockholders.
Plastic blockholders are preferable.
4. Polymerize at room temperature for 2-4 hr. The system is less sensitive to inhibition by atmospheric oxygen, so sealing blockholders with paraffin is not necessary. Further polymerization for an additional 2 hr at
37C may be advisable.
5. After polymerization remove the blocks from the
molds and cut 1-5 p m sections.
6. Stretch sections on distilled water and mount them on
clean slides. Dry sections at room temperature or on a
hot plate at 60C.

C. Standard Processing Schedule for

Zrnmunohistochemi~tryUsing RES G20 (Technovit 8100)
1. Fix thin tissue slices, of maximum size 10 x 10 x 1
mm, by immersion in 2% (wlv) formaldehyde in
phosphate buffered saline (PBS), pH 7.4, for 3 4 hr
at 4C. Prepare the formaldehyde from paraformaldehyde according to Karnovsky (59).
2. Rinse the tissue slices in 0.1 M phosphate buffer pH
7.4 or in PBS containing 6.8% sucrose, overnight at
4C.
3. Dehydrate the tissue in acetone absolute 30-60 min.
at 4C. Change the acetone repeatedly until the solution remains clear.
4. Infiltrate in RES G20JTechnovit 8100 Solution A
(Table 2) for 8-10 hr at 4OC.
5. Following infiltration, place the tissue slices into the
embedding molds containing the embedding Solution C (infiltration Solution A to which the accelerator solution B is added (Table 2). Note: Bring the
GMA Embedding for Light Microscopy 1 Gerrits and Horobin

Downloaded by [Monash University Library] at 18:24 15 April 2016

tissues in contact with Solution C for 10 min prior to

placing them in the embedding molds.
6. Place the blockholders into the embedding molds,
and pour melted paraffin (60C) around the blockholders to exclude atmospheric oxygen.
7. Polymerize in a refrigerator on cracked ice (OC),
overnight.
8. Following polymerization, store the resin blocks at
20C.

The Journal of Histotechnology 1 Vol. 19, No. 4 1 December 1996

9. Remove the blocks from the molds and cut 2 F m

thick sections. It is advisable to cut the sections under
controlled, standardized conditions in a temperature
controlled room (20C), at 60% relative humidity.

10. Stretch sections on distilled water (20C) and mount

them on glass slides.
1I . Dry sections at 37C for 2 hr, and store at 4C. Stain
sections for imrnunohistochernistry. It is advisable to
use modifications according to van Goor et a1 (90).