258
7.
"STAINING"
about 11 usually can be expected to be much more effective than when
used near neutrality or in acid solution. The easiest way to achieve such
alkalinity is to dissolve the stain in a dilute borax solution rather than
water. Of course not all stains can be handled in this way.
Alcoholic stains can be expected to penetrate cross-linked plastics
better than aqueous ones, and should be used when possible.
Stains with the reputation for acting so powerfully that overstaining
with conventional material is a problem, and are ordinarily differentiated
by destaining, are apt to be most effective with material embedded in
epoxy or polyester plastics. Furthermore they may be effective without
differentiation with thinner sections than would otherwise be possible.
The complaint of conventional microscopists in viewing even wellstained sections of the electron microscopists is that they tend to lack
dramatic contrast. To be sure, if osmium tetroxide fixation was employed,
probably there is a gray background tone [which, however, may be
somewhat bleached by prolonged soaking in dioxane or, better, by pretreatment with peracetic acid reagent as recommended by Munger (1961);
see below]. More importantly, though, the excellent tissue preservation
that the electron microscopist achieves retains so much material, so
uniformly fixed, that there are not the major discontinuities which
exaggerate contrast in conventional microscopy. So much material remains everywhere to be stained, that there are not apt to be dramatic
differences in stain intensity, and one differentiates different structures
mainly with color. Actually the conventional light microscopist can see
all of the details that he is used to, and more, if he has a well-stained
section about 0.5-1 u. thick. If he appreciates that he is seeing a better
preserved tissue than he has ever looked at before, his experience will be
more meaningful. It should also be indicated that it is often profitable
to look at sections much less than 1 micron thick, for detail can be seen in
such sections with unparalleled resolution, particularly with phase microscopy. It is almost impossible to stain such sections intensely, however,
simply because there is not enough material present in the unusually thin
sections to bind a sufficient quantity of dye for great brilliance. In such
circumstances, as indicated above, it is probably best to use dyes which,
by conventional standards, easily overstain. Then they can be employed
with thin sections at full strength for maximum possible brilliance.
A part of the bad reputation for difficult stainability the epoxy
and polyester plastics have acquired surely derives from the fact that
ordinarily hematoxylin and acid dye counterstains prove to be almost
7.
"STAINING"
about 11 usually can be expected to be much more effective than when
used near neutrality or in acid solution. The easiest way to achieve such
alkalinity is to dissolve the stain in a dilute borax solution rather than
water. Of course not all stains can be handled in this way.
Alcoholic stains can be expected to penetrate cross-linked plastics
better than aqueous ones, and should be used when possible.
Stains with the reputation for acting so powerfully that overstaining
with conventional material is a problem, and are ordinarily differentiated
by destaining, are apt to be most effective with material embedded in
epoxy or polyester plastics. Furthermore they may be effective without
differentiation with thinner sections than would otherwise be possible.
The complaint of conventional microscopists in viewing even wellstained sections of the electron microscopists is that they tend to lack
dramatic contrast. To be sure, if osmium tetroxide fixation was employed,
probably there is a gray background tone [which, however, may be
somewhat bleached by prolonged soaking in dioxane or, better, by pretreatment with peracetic acid reagent as recommended by Munger (1961);
see below]. More importantly, though, the excellent tissue preservation
that the electron microscopist achieves retains so much material, so
uniformly fixed, that there are not the major discontinuities which
exaggerate contrast in conventional microscopy. So much material remains everywhere to be stained, that there are not apt to be dramatic
differences in stain intensity, and one differentiates different structures
mainly with color. Actually the conventional light microscopist can see
all of the details that he is used to, and more, if he has a well-stained
section about 0.5-1 u. thick. If he appreciates that he is seeing a better
preserved tissue than he has ever looked at before, his experience will be
more meaningful. It should also be indicated that it is often profitable
to look at sections much less than 1 micron thick, for detail can be seen in
such sections with unparalleled resolution, particularly with phase microscopy. It is almost impossible to stain such sections intensely, however,
simply because there is not enough material present in the unusually thin
sections to bind a sufficient quantity of dye for great brilliance. In such
circumstances, as indicated above, it is probably best to use dyes which,
by conventional standards, easily overstain. Then they can be employed
with thin sections at full strength for maximum possible brilliance.
A part of the bad reputation for difficult stainability the epoxy
and polyester plastics have acquired surely derives from the fact that
ordinarily hematoxylin and acid dye counterstains prove to be almost