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EMBEDDING METHODS
JOHN H. L U F T , M.D.
ABSTRACT
Epoxy embedding methods of Glauert and Kushida have been modified so as to yield rapid,
reproducible, and convenient embedding methods for electron microscopy. The sections
It has become apparent that there is considerable the recommended times, and (2) difficulties in
advantage in using epoxy resins in embedding cutting, resulting in corrugated or shattered sec-
tissues for electron microscopy. Compared to tions, even after prolonged soaking in the liquid
methacrylates, epoxy resins offer remarkable resin in attempts to achieve adequate impregna-
freedom from polymerization damage, with con- tion.
sequent excellent preservation of cellular fine These difficulties have been apparent in other
structure and intercellular relationships (Birbeck laboratories as well, and have led to the develop-
and Mercer, 1956). Epoxy sections also seem to ment of embedding methods based on other
suffer less degradation during irradiation by the epoxy resins. Thus Kushida (1959) and Finck
electron beam than do those cut from methacry- (1960) have published methods using an ali-
late. They can be used with many fixatives without phatic epoxy resin marketed by the Shell Chemical
danger of "bubbling." Corporation under the name, Epon 812 (previ-
The first practical method of embedding in ously known as Epon 562). These methods have
epoxy resins was that of Glauert et al. (1956, not been tried in this laboratory.
1958). This procedure has by now received a con- Quite independently, over the last three or
siderable trial, and has yielded very fine results in four years, there have been developed in this
the hands of some investigators. laboratory two separate approaches to epoxy
On the other hand, we have had difficulty in embedding. These have had sufficient success to
using these resins, even with the British materialsx warrant publication. The first approach involved
said to be identical to those recommended by use of the aromatic Araldite resins of the Ciba
Glauert et al. (1956, 1958). The use of so called Company, adapting these materials to yield
"equivalent" materials of United States origin rapid, reliable procedures of embedding. The
has given even more trouble. Problems encoun- second approach was similar in some respects to
tered with these Araldites involve (1) inadequate that of Kushida (1959) and Finck (1960). It is
penetration of tissue blocks (1 to 1.5 mm. cubes) in likewise based on aliphatic Epon 812 and has led
to rapid, flexible, versatile, and reliable methods
1 The author is indebted to Dr. H. Huxley, Uni-
of embedding which have become routine in this
versity College London, to Professor J. T. Randall,
King's College, London, to Dr. M. S. C. Birbeck, laboratory. These Epon methods have been sent to
Chester Beatty Research Institute, London, and to several laboratories in other cities, where they
Mr. H. Voorhees, Ciba, Inc., Kimberton, Pennsyl- have been applied successfully. They are now
vania, for gifts of the British materials; i.e., Araldite used in our laboratories for instruction of beginners
casting resin M, hardener 964B, and accelerator in biological electron microscopy. Students be-
964C. ginning with Epon embedding methods have, in
409
our experience, achieved satisfactory specimens in MODIFIED ~ARALDITE'' METHOD
a m u c h shorter time than was necessary when
methacrylate was used. M e t h a c r y l a t e has been T h e main defect of Glauert's Araldite, that ot slow
a b a n d o n e d in our laboratories as an e m b e d d i n g penetration, was ameliorated by using propylene
m e d i u m for electron microscopy. oxide as an additional stage in the transition be-
T h e new Araldite and E p o n procedures have led tween the d e h y d r a t i n g alcohols a n d the liquid
to significant improvements in case of penetration resin. Various reactive diluents are available to
a n d of cutting, as c o m p a r e d lo Glauert and reduce the high viscosity of the liquid epoxy
Glauert's (1958) Araldite methods. For most tissues, resin mixture, but most of these tend to degrade
the new methods permit e m b e d d i n g of tissue the mechanical properties of the cured, solid
cubes from fixation to sectioning in 24 to 48 hours. resins. (Non-reactive diluents, such as xylene, etc.,
T h e blocks can be cut nearly as easily as can the are even worse in this respect and may even sepa-
methacrylates. W i t h the Epon resin the hardness rate from the resin during polymerization). A
can be readily varied over a wide range. In the series of mono-epoxides was tried as reactive
electron microscope, Epon sections yield better diluents at the ratio of 10 per cent v/v in the final
contrast than Araldite preparations. Either Epon resin. T h e mono-epoxides used in these trials
or Araldite sections can be stained readily, either were C,6-Cls olefin oxide, dodecene oxide, di-
410 TIlE JOURNAL OF BIOPHYSICAL AND BIOCIIEMICAL CYTOLOGY " VOLUME ,0, 1961
in the highly reactive Epon resin. In this case, parting of the section from the block at the ad-
considerable extraction of the P T A occurs. Acids vancing molten or softened region.
in general, and P T A in particular, are reactive M a n y of the attractive features of epoxy resins
with epoxides (Lee and Neville, 1957); in the case for industrial applications result from the mechani-
of solid P T A and pure propylene oxide, the reac- cal properties associated with a high degree of
tion is explosive. Staining of the tissue block can cross-linkage, which can be obtained with the
be preserved by removing the excess protons from formulations recommended for commercial use.
the bound P T A with alkali, as follows: Following These highly cross-linked resins prove to be very
the usual P T A treatment and a 30-minute abso- difficult to cut. Epoxy resins cured by mono-
lute alcohol rinse, the tissue blocks can be exposed anhydrides are chemically less subject to such
for 15 to 30 minutes to 0.01 per cent N a O H in cross-linkage than are resins cured by other
absolute alcohol followed by a 30-minute rinse in means. Moreover, according to Lee and Neville
two changes of absolute alcohol. The tissue may (1957), the amount of cross-linkage can be modi-
then be run into propylene oxide and embedded fied further to some extent by varying the tem-
by one of the methods described here. perature of the curing cycle. Thus low tempera-
The second problem, namely difficulty in sec- tures are said to favor a linear polymer with few
tioning, has been lessened by two alterations in the cross-linkages. Lee and Neville state further that
TABLE I
Total Accelerator
Mixture A Mixture B volume (DMP-30)
(volume) (volume) resin (volume) Equivalent hardnessas related to methacrylates