126
4.
EMBEDDING
stances from tissues, but also less tendency to disrupt the native structures of proteins and other macromolecules. Normal antigenic activity
is known to be retained intact in certain instances, and thus sections in
this embedment can be treated with ferritin-antibody conjugates which
then find their appropriate targets (Chapter 7.10).
In essence, McLean and Singer (1964) have found they could cross-link
an anionic monomer with a cationic one in any reasonable proportion,
so that the net charge could be near zero, or so that the final plastic
could be decidedly acidic or basic in its characteristics. When a neutral
plastic was desired, a 1:1 molar mixture of the anionic methacrylic acid
(MA) was prepared with cationic
dimethylaminoethylmethacrylate
(DMA). When these substances were mixed, considerable heat was
evolved so, to prevent thermal polymerization, MA was added by drops
with stirring to the DMA. A 0.25 M quantity of the cross-linking monomer, tetramethylenedimethacrylate (TMA) was also added, and finally
0.025% of the catalyst, azodiisobutyronitrile. When the authors wished
a decidedly negative charge in the completed embedment, the final molar
mixture of monomers was altered to 2(MA): l(DMA): 0.33(TMA). Rohm
and Haas, Philadelphia 5, Pennsylvania, supplied the MA and DMA;
Monomer-Polymer Labs, Inc., Philadelphia 25, Pennsylvania, was the
source of the TMA; and Eastman Chemicals, Rochester 3, New York, sold
the catalyst. The monomer of MA was further purified by vacuum distillation at 10 mm pressure. The fraction boiling at 60°C was collected
and stored at 4°C. The other monomers tended to polymerize on distillation, and therefore were used as supplied.
Aldehyde-fixed tissue, previously washed in pure buffer, was passed
through successive concentrations of the monomer mixture at 4°C until
the water was removed. Tissue was finally soaked for 12 hours at 4°C in
several changes of the embedding medium before transferring to freshly
prepared embedding medium in gelatin capsules. Polymerization was
accomplished by fluorescent ultraviolet radiation for 2 days, also at 4°C.
Because of the hydrophilic nature of the embedment, some difficulties
were encountered in sectioning. The block face was particularly prone
to wetting. A maximally acute knife angle is recommended with a
relatively fast cutting speed. A trough filled only with water seemed to
work best. Sections finally were collected on unusually fine grids (1000
mesh) without a supporting film. Staining apparently was unnecessary
for most observation.
4.
EMBEDDING
stances from tissues, but also less tendency to disrupt the native structures of proteins and other macromolecules. Normal antigenic activity
is known to be retained intact in certain instances, and thus sections in
this embedment can be treated with ferritin-antibody conjugates which
then find their appropriate targets (Chapter 7.10).
In essence, McLean and Singer (1964) have found they could cross-link
an anionic monomer with a cationic one in any reasonable proportion,
so that the net charge could be near zero, or so that the final plastic
could be decidedly acidic or basic in its characteristics. When a neutral
plastic was desired, a 1:1 molar mixture of the anionic methacrylic acid
(MA) was prepared with cationic
dimethylaminoethylmethacrylate
(DMA). When these substances were mixed, considerable heat was
evolved so, to prevent thermal polymerization, MA was added by drops
with stirring to the DMA. A 0.25 M quantity of the cross-linking monomer, tetramethylenedimethacrylate (TMA) was also added, and finally
0.025% of the catalyst, azodiisobutyronitrile. When the authors wished
a decidedly negative charge in the completed embedment, the final molar
mixture of monomers was altered to 2(MA): l(DMA): 0.33(TMA). Rohm
and Haas, Philadelphia 5, Pennsylvania, supplied the MA and DMA;
Monomer-Polymer Labs, Inc., Philadelphia 25, Pennsylvania, was the
source of the TMA; and Eastman Chemicals, Rochester 3, New York, sold
the catalyst. The monomer of MA was further purified by vacuum distillation at 10 mm pressure. The fraction boiling at 60°C was collected
and stored at 4°C. The other monomers tended to polymerize on distillation, and therefore were used as supplied.
Aldehyde-fixed tissue, previously washed in pure buffer, was passed
through successive concentrations of the monomer mixture at 4°C until
the water was removed. Tissue was finally soaked for 12 hours at 4°C in
several changes of the embedding medium before transferring to freshly
prepared embedding medium in gelatin capsules. Polymerization was
accomplished by fluorescent ultraviolet radiation for 2 days, also at 4°C.
Because of the hydrophilic nature of the embedment, some difficulties
were encountered in sectioning. The block face was particularly prone
to wetting. A maximally acute knife angle is recommended with a
relatively fast cutting speed. A trough filled only with water seemed to
work best. Sections finally were collected on unusually fine grids (1000
mesh) without a supporting film. Staining apparently was unnecessary
for most observation.