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Joiirnal of Microscopy, Vol. 132, Pr 1, Ocrober 1983, p p . 81-85.

Received 9 Septeitrber 1982; acrepred 12 . ~ ~ t r r i t u r1983


y

A new, less toxic polymerization system


for the embedding of soft tissues in glycol methacrylate
and subsequent preparing of serial sections
by P. 0. G E R R I Tand
S L. SMID*, Department of Anatomy and Embryology, University of
Groningen, Groningen, and * Stichting Samenwerking Dewtse Ziekenhuizen, Derf, The Netherfands
K E Y WORDS.

Glycol methacrylate, GMA, plastic, softener, polymerization, serial sectioning.

SUMMARY

The paper describes a new polymerization system for embedding soft tissues in glycol
methacrylate (GMA). T h e polymerization of GMA is initiated by means of a barbituric acid
derivative in combination with chloride ions and dibenzoyl peroxide. The catalyst system
contains no aromatic amines which constitutes a toxicological advantage over the commonly
employed system of peroxide 'aromatic amine. Clear blocks are obtained from which 1-2 km
sections are easy to cut. I n combination with an appropriate softener, polyethylene glycol 400,
serial sectioning may be practised.
INTRODUCTION

Since the introduction of glycol methacrylate (GMA) as an embedding medium in light


microscopic research (Ashley & Feder, 1966; Ruddell, 1967), this medium has become increasingly popular. The method has many inherent advantages : well-preserved morphology and
easy thin sectioning in the range of 1-4 pm. Tissue shrinkage and distortion are minimal and
may be controlled by working undcr standardized conditions (Hanstede & Gerrits, 1983).
However, in contrast to the paraffin embedding techniques, serial sectioning and obtaining
ribbons from GMA embedded tissues seems to be rather problematical. GMA sections have to
be picked up one by one from the knife edge with a pair of twcczcrs.
As far as we know, only one article, by Engcn & Wheeler (1978), describes a technique in
which a combination of N-butyl methacrylate and paraffin makes serial sectioning possible.
In our laboratories we roucinely apply E F L 67 (Suva), JBi (Polysciences Inc.) and our own
mixtures (Hanstede & Gerrits, 1982, 1983), but the toxicity of the tertiary amine accelerator
system, N,N-dimcthylaniline, remains a major disadvantage.
This papcr describes a hydrophilic embedding material based on water-soluble GMA,
combined with a non-toxic catalyst system. T h e combination of this catalyst system (Bredereck
et af., 1966; Gross, 1977) and the plasticizer polyethylene glycol 400 (PEG 400) enables serial
sectioning of soft tissues ernbedded in GMA.
MATERIALS AND METHODS

Pieces of rat liver, kidney, spleen, lung and tongue were fixed by immersion or by perfusion
in neutralized 4" formaldehyde, prepared from paraformaldehyde (Aldrich Chem. Comp.
Inc.) according to the method of Karnovsky (1965). After adequate fixation, small pieces of
2 x 4 x 10 mm were excised and dehydrated using ascending concentrations of ethanol starting
with ethanol 70",,. Although GMA is miscible with water a pre-infiltration step in a solution
,)

('1

1983 The Royal Microscopical Society

81

82

P. 0. Gerrits and L. Smid

consisting of absolute alcohol: infiltrating solution in equal amounts was used in this procedure
as the final step prior to complete infiltration. Tissue was placed in the infiltrating solution and
left up to 24 h, depending on the size of the tissue.
Composition of solutions employed in the embedding procedure
A. Infiltrating solution: Tcchnovit 7100
40 ml
(glycol methacrylate, containing Co-catalyst XCl)
Polyethylene glycol 400
4 ml
Dibenzoyl peroxide
0.5 g
(moistencd with 20",, H.0)
15 ml
B. Embedding solution : Infiltrating solution A
Technovit 7100, hardener I1
1 ml
(the accelerator solution, containing a barbituric acid
derivate)
After infiltration the tissues were placcd in Teflon ernbedding moulds (Histoform S, Fa.
Kulzer & Co. GmbH) or Sorvall ' moulding cups fitted with aluminium blockholdcrs (Du Pont,
Biomedical Division). Approximatcly 1 ml of embedding solution B is poured into each form.
Subsequently, each piece of tissue should be rcpositioned within the embedding form in such a
way as to ensure complete bubble-frec cnclosure of the tissue by the ernbedding solution. The
polymerization was completed at room temperature within 2 h. Further polymerization for an
additional 2 h at 310 K is recommended.
T o determine the effect of softener on sectioning quality a further five softeners (butoxyethanol, cyclohexanol, dibutyl phtalate, ethylene glycol and polyethylene glycol 600) in the same
concentration as P E G 400 werc tested.
Sectioning was done by means of a 'Jung 1140 autocut' microtome provided with a D-knife
with tungsten carbide cutting edge. 1 or 2 pm thick sections were cut by means of the autodrive
or by hand. This took place under controlled conditions at room tcmperaturc (293 K) and a
relative humidity of 60",,. After stretching the sections on a waterbath containing distilled
water at room tcmperature, the stretching and drying procedure was completed on a hot plate
at 333 K.
Details of section handling arc described in the papers of Bennett et al. (1976) and Hanstede
& Gerrits (1982). Horizontal blockrims parallel to the cutting edge are critical when ribbons have
to be made.
Suppliers for all employed materials: all chemicals as well as PTFE-histoform S: Kulzer &
Co. GmbH, Im Dammwald 21, D-6382 Friedrichsdorf 1, G.F.R. ; Sorvall PE-molding cuptray
No. 45 459: D u Pont Co., Biomedical Division, Ncwtown Connccticut 06470, U.S.A.; softeners:
Fluka AG, Chcmische Fabrik, CH-9470 Buchs, Switzerland.
RESULTS AND DISCUSSION

GMA combined with P E G 400, when polymerized in the way described above, is outstanding in view of the many advantages it shows over the GMA-bcnzoyl peroxide N,N-dimcthylTable 1. T h e influence of softcncrs upon the sectioning quality of tissues embedded in Technovit 7100.
All the softeners were tested and compared to each other in the same Concentration, not exceeding lo",,
by weight GMA.
Softener
Butoxyethanol
Cyclohexanol
Dibutylphtalate
Ethylene glycol
Polyethylene glycol 400
Polyethylene glycol 600

Sectioning
quality

++
++
+

Rcmarks

A milky block, and curling


of the sections

+ + t

+++
-

Ribbons
A brittle block

Soft tissue embeddinq in glycol methacrylate

83

aniline system: a compilation of less toxic compounds, a plastic block of uniform hardness that
stays clear even after many years.
By measuring the maximum temperatures occurring within the blocks during the polymerization process it could be shown that a temperature of 313 K was never exceeded. The
polymerization system is less sensitive to inhibition by oxygen, and pouring paraffin around the
block holder in order to exclude air is not necessary.
The described Technovit 7100 embedding system is based on the water-soluble GMA to
which chloride ions (Co-catalyst XCl) are added. In combination with benzoyl peroxide and
the accelerator (Technovit 7100, hardener II), which contains a barbituric acid derivate the
polymerization can be initiated and promoted.

Fig. 1. 2 prn serial sectioning of kidney embedded in glycol methacrylate.

The ultimate hardness of the entire block can be determined by varying the amount of the
softener PEG 400 (limit: not exceeding lo",, by weight). In our experiments we have evaluated
several softeners such as 2-butoxycthanol, cyclohexanol, dibutyl phtalate, ethylene glycol,
PEG 400 and PEG 600. Table 1 shows the influence of softeners upon GMA sectioning quality.
In our opinion the manufacturing of section ribbons is possible due to a combined action of
the barbituric acid polymerization system and the softener PEG 400 (Fig. 1). Good sectioning
results were also obtained using ethylene glycol in the same concentration as PEG 400. Soft
tissue sections (1-2 pm) prepared by our method were tested for routine staining and histochemistry. The sections were stained without removing the embedding medium. Routine
staining: Gill's haematoxylin-eosin and a haematoxylin and eosin-like stain according to Troyer
& Babich (1981).
Histochemistry : periodic acid-Schiff (PAS), periodic acid methenamine silver (PAMS),
toluidi ne blue, Feulgen and Prussian blue (Perk).
The sections are of excellent quality (Fig. 2) and show fine morphological detail equal to
sections from traditional embedding systems. Staining sections yields no problems at all and
we have the experience that only minimal modifications are required in order to obtain optimal
staining results.

84

P. 0. Gerrits and L. Smid

Fig. 2

Soft tissue embedding in glycol methacrylate

85

ACKNOWLEDGMENTS

We wish to thank Mrs J. G. Bcnjamins, Mrs G. T. Hoogenberg, A. J. H. Deddens and


R. A. Wieringa for their assistance in the preparation of the manuscript, D r R. N. J. Cupkdo
for his critical reading and helpful comments and Kulzer Sr Co. for their technical assistance
and all supplied materials.
REFERENCES

Ashley, C.A. & E'eder, N. (1966) Glycol methacrylate in histopathology. Arch. Parhol. 81, 381.
Bennett, H.S., Wyrick, A.D., Lee, S.W. & McNcil, J.H., Jr (1976) Science and Art in preparing tjssues
ernbedded in plastic for light microscopy, with special reference to glycol methacrylate, glass knives
and simple stains. S t a i n Teclinol. 51, 71.
Bredereck, H., Petranyi, P., Possclt, K . 8( Sigel, H. (1966) Uber CH-aktive Polymerisations Initiatoren,
XIII. Mitt. Polymerisationen und Polymerisationsinitiatoren. Makronrol. Chew. 92, 70.
Engen, P.C. & Wheeler, R. (1978) N-Butyl methacrylate and paraffin as an embedding medium for light
microscopy. Sruin Techtiol. 53, 17.
Gross, A. (1977) 1st die Verwcndung von Kaltpolymcrisat bei der Herstellung dcfinitiver Prcthesen nach
dcm heutigen Entwicklungsstand zu vertreten ? Qitintessenz d . Zahntechnik. 7 , 45.
Hanstede, J.G. Ik Gerrits, P.O. (1982) A new plastic for morphomctric investigation of blood vcsscls,
especially in large organs such as the human liver. Anar. Rec. 203, 2.
Hanstede, J.G. & Gcrrits, P.O. (1983) T h e effects of ernbedding in water-soluble plastics on the final
dimensions of liver sections. .7. Microzc. 131, 79.
JB-4 Embedding Kit T M (1976) Data sheet 123, Polysciences Inc.
Karnovsky, M .J. (1965) A formaldehydc-glutaraldehydefixative of high osmolarity for use in electron
microscopy. .7. Cell 13iol. 27, 137A.
Ruddell, L.C. (1967) Hydroxyethyl methacrylate combined with polyethvlene glycol 400 and water; an
ernbedding medium for routine 1-2 micron sectioning. Sraiti Teclinol. 42, 119.
Troyer, H. & Babich, E. (1981) A hacmatoxylin and eosin-like stain for glycol methacrylate embedded
tissue sections. Stain Technol. 56, 39.

Fig. 2. (a) Prussian blue reaction according to Perk, showing haemosiderin pigments in the cells of the
red spleen pulp. Nucleus counterstained with Safranin 0; 2 pm, x 500. (b) Section of foliate papilla of
rat tongue. Gill's haematoxylin and eosin: 2 pm, x 125. (c) Periodic acid methenamine silver staining of
rat kidney, basement membranes of glomerulus and renal tubule are selectively stained and defined as
fine linear structures; 2 pm, x 500. (d) Rat hepatic parenchymal cells with glycogen deposits, periodic
acid-Schiff (PAS). Nucleus counterstained with Gill's haematoxylin ; 2 /mi, x 500.

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