7.11.
SPECIFIC ANTIBODY "STAINS"
249
was specific for tobacco mosaic virus, and antigen-antibody precipitations
simply were prepared in bulk, and sprayed upon coated grids (Chapter
10.2) for examination in the electron microscope. Subsequent work by
other investigators often has been concerned with attaching the antibody to its antigen in living cells before fixation, or to cells after fixation,
and then preparing such cells or tissues for electron microscopy by conventional means. There is also the hope that antibodies could find their
specific targets after sections had been prepared, which would afford an
exceptionally favorable approach to the study of intracellular antigens.
Of course the use of specific antibodies with preserved cells presupposes
that fixation does not destroy the combining capacity of the antigens in
question. Probably most satisfactory from this point of view are aldehydes
which are known in several specific cases not to have a deleterious effect.
There are no particular problems in introducing antibodies into living
cell suspensions although they cannot be expected to penetrate cell interiors. Tissues pose more serious difficulties. However, Arhelger, Gronvall, Carr, and Brunson (1963) successfully coupled conjugated antibodies
within 1 mm
3
cubes of isolated living tissue with only a 3 minute exposure in a buffered salt solution. The tissue blocks then were washed
briefly and fixed. These authors report trying to give coupled antibodies
to whole animals but without success.
Singer and McLean (1963) cite two methods that have been used to
render cell membranes permeable to ferritin coupled antibodies for "preembedding staining." With bacterial cells, lysozyme has been used, and
with cultured animal cells, freezing and thawing permitted access. But
surely other possibilities exist, particularly when dealing with fixed
material.
"Post-embedding staining" encounters several serious technical obstacles. It requires the antigenic determinant to retain its combining
capacity, not only through fixation, but also in the more or less reactive
solvents used along the way to embedment. It is usually expected that
proteins change their physico-chemical character radically in nonaqueous
solvents, a change which well might inactivate protein antigens. Highly
reactive epoxy monomers are particularly suspect, but fortunately there
are both practical and theoretical reasons for thinking that methacrylate
plastics may not prove too seriously damaging. A further technical obstacle to staining sections is the finding that ferritin-antibody conjugates
are nonspecifically and irreversibly absorbed on methacrylate, glycol
methacrylate, Epon and Vestopal. However, some progress has been made
SPECIFIC ANTIBODY "STAINS"
249
was specific for tobacco mosaic virus, and antigen-antibody precipitations
simply were prepared in bulk, and sprayed upon coated grids (Chapter
10.2) for examination in the electron microscope. Subsequent work by
other investigators often has been concerned with attaching the antibody to its antigen in living cells before fixation, or to cells after fixation,
and then preparing such cells or tissues for electron microscopy by conventional means. There is also the hope that antibodies could find their
specific targets after sections had been prepared, which would afford an
exceptionally favorable approach to the study of intracellular antigens.
Of course the use of specific antibodies with preserved cells presupposes
that fixation does not destroy the combining capacity of the antigens in
question. Probably most satisfactory from this point of view are aldehydes
which are known in several specific cases not to have a deleterious effect.
There are no particular problems in introducing antibodies into living
cell suspensions although they cannot be expected to penetrate cell interiors. Tissues pose more serious difficulties. However, Arhelger, Gronvall, Carr, and Brunson (1963) successfully coupled conjugated antibodies
within 1 mm
3
cubes of isolated living tissue with only a 3 minute exposure in a buffered salt solution. The tissue blocks then were washed
briefly and fixed. These authors report trying to give coupled antibodies
to whole animals but without success.
Singer and McLean (1963) cite two methods that have been used to
render cell membranes permeable to ferritin coupled antibodies for "preembedding staining." With bacterial cells, lysozyme has been used, and
with cultured animal cells, freezing and thawing permitted access. But
surely other possibilities exist, particularly when dealing with fixed
material.
"Post-embedding staining" encounters several serious technical obstacles. It requires the antigenic determinant to retain its combining
capacity, not only through fixation, but also in the more or less reactive
solvents used along the way to embedment. It is usually expected that
proteins change their physico-chemical character radically in nonaqueous
solvents, a change which well might inactivate protein antigens. Highly
reactive epoxy monomers are particularly suspect, but fortunately there
are both practical and theoretical reasons for thinking that methacrylate
plastics may not prove too seriously damaging. A further technical obstacle to staining sections is the finding that ferritin-antibody conjugates
are nonspecifically and irreversibly absorbed on methacrylate, glycol
methacrylate, Epon and Vestopal. However, some progress has been made