118
4.
EMBEDDING
Vestopal has a fine grain. Sections can be stained easily, certainly more
easily than Araldite ones. It sections nicely, although it is sufficiently
hard so that only small sections can be obtained. It decomposes slightly
during electron bombardment, but without damaging the specimen.
Thus its density is slightly reduced, and image contrast is improved.
Vestopal is made by the Chemische Werke, Huls, but is available in small
quantities along with the activator and initiator from Martin M. Jaeger,
Vesenaz, Geneva, Switzerland. Ryter and Kellenberger prefer tertiary
butyl perbenzoate as the initiator, and cobalt naphthenate as the activator. They warn that these substances must not be mixed directly together for the combination is explosive. These substances are said not
to keep well, even in a refrigerator and protected from light, and must
be replaced every few months. This seems to be an undue caution for we
have successfully used these compounds after keeping them in refrigerated storage for over a year, and we have used activator after it had become very darkly discolored indeed.
Vestopal is not miscible with alcohol. Therefore, it was recommended
originally that dehydration be concluded with anhydrous acetone, from
which it was passed through a graded series of Vestopal and acetone mixtures, allowing 30-60 minutes in each. Because of some difficulty in getting anhydrous acetone (Chapter 4.3), we originally substituted propylene oxide. However, this evaporates so rapidly that condensation is a
problem. It seems evident that styrene is the intermediate solvent of
choice for it is a "reactive" solvent. Its use was first proposed by Kurtz
(1961). Tissue is finally infiltrated with pure Vestopal plus 1% of the
"initiator" and 1% of the "activator." Specimens are left in this for 30-60
minutes, and then transferred to capsules with a fresh mixture. The capsules are polymerized at 60°C overnight, or for 24 hours. In case softer
blocks are desired, dibutyl phthalate may be added as a plasticizer in
concentrations up to 10%. This is not to be recommended in general,
however.
Vestopal mixtures, complete with activator and initiator, can be stored
for many months in a refrigerator without evident deterioration. Although it might be safer to freeze mixtures, our own machine, operating
at 1°-3°G has proved adequate. Plastic mixtures have been divided into
relatively small glass jars so that their contents are used fairly quickly
once they are opened. Of course the jars must be warmed to room temperature before opening to avoid water condensation. It is safe to rechill
the mixtures several times. Unfortunately, disposable polystyrene syringes
4.
EMBEDDING
Vestopal has a fine grain. Sections can be stained easily, certainly more
easily than Araldite ones. It sections nicely, although it is sufficiently
hard so that only small sections can be obtained. It decomposes slightly
during electron bombardment, but without damaging the specimen.
Thus its density is slightly reduced, and image contrast is improved.
Vestopal is made by the Chemische Werke, Huls, but is available in small
quantities along with the activator and initiator from Martin M. Jaeger,
Vesenaz, Geneva, Switzerland. Ryter and Kellenberger prefer tertiary
butyl perbenzoate as the initiator, and cobalt naphthenate as the activator. They warn that these substances must not be mixed directly together for the combination is explosive. These substances are said not
to keep well, even in a refrigerator and protected from light, and must
be replaced every few months. This seems to be an undue caution for we
have successfully used these compounds after keeping them in refrigerated storage for over a year, and we have used activator after it had become very darkly discolored indeed.
Vestopal is not miscible with alcohol. Therefore, it was recommended
originally that dehydration be concluded with anhydrous acetone, from
which it was passed through a graded series of Vestopal and acetone mixtures, allowing 30-60 minutes in each. Because of some difficulty in getting anhydrous acetone (Chapter 4.3), we originally substituted propylene oxide. However, this evaporates so rapidly that condensation is a
problem. It seems evident that styrene is the intermediate solvent of
choice for it is a "reactive" solvent. Its use was first proposed by Kurtz
(1961). Tissue is finally infiltrated with pure Vestopal plus 1% of the
"initiator" and 1% of the "activator." Specimens are left in this for 30-60
minutes, and then transferred to capsules with a fresh mixture. The capsules are polymerized at 60°C overnight, or for 24 hours. In case softer
blocks are desired, dibutyl phthalate may be added as a plasticizer in
concentrations up to 10%. This is not to be recommended in general,
however.
Vestopal mixtures, complete with activator and initiator, can be stored
for many months in a refrigerator without evident deterioration. Although it might be safer to freeze mixtures, our own machine, operating
at 1°-3°G has proved adequate. Plastic mixtures have been divided into
relatively small glass jars so that their contents are used fairly quickly
once they are opened. Of course the jars must be warmed to room temperature before opening to avoid water condensation. It is safe to rechill
the mixtures several times. Unfortunately, disposable polystyrene syringes