2.6.
VASCULAR PERFUSION
23
an organ is transected. This, of course, will depend upon the organ, but
much more flushing with fixative may be required to seal the surface than
if a natural surface is used. Cotton swabs may be used to control the first
more or less massive bleeding, followed by vigorous flushing with fixative.
Avoid the use of salt solutions for this purpose. In general, success with
this method will depend upon anatomically avoiding the larger blood
vessels so that massive hemorrhages do not occur.
Once again it is pertinent to stress that the eyes must be protected
from the fumes of osmium tetroxide, for under the pressure of working
efficiently and fast, it is all too easy to forget about one's eyes until
corneal damage has been done.
2.6. Vascular Perfusion
It is natural for one acquainted with the techniques of conventional
microscopy to think of vascular perfusion as a likely way of preserving
tissue for electron microscopy. This can be done very successfully when
buffered aldehyde fixatives are used, but cannot be done without heroic
means and extraordinary attention to detail with osmium tetroxide
fixation.
We have had a good deal of experience using buffered formaldehyde
(Chapters 3.9 and 3.10) as a perfusing fluid. Ordinarily we have used it as a
killing agent and primary fixative to gain time to retrieve deeply placed
tissue, in order to do careful dissections and to expose anatomically difficult
tissue, which then was fixed secondarily with osmium tetroxide. It is
ideal for this purpose, and makes it possible to collect well-fixed tissue
that would be difficult indeed to obtain in any other way. Furthermore, it
can be used to harden particularly soft tissues so that suitably small
blocks then can be prepared without mechanical damage for secondary
fixation with osmium tetroxide.
Our perfusion apparatus is commonplace and simple. It relies upon
gravity flow from a fixative bottle about 4 ft above the operating level.
The tube from this usually terminates in a 3-way stopcock of the type
used with syringe needles. Very large caliber syringe needles (for rats, 16
gage; for cats, 12 gage) then serve as cannulae. These usually are deliberately blunted, particularly when the perfusion is expected to be
directly into a heart ventricle. This whole system is filled with fixative in
VASCULAR PERFUSION
23
an organ is transected. This, of course, will depend upon the organ, but
much more flushing with fixative may be required to seal the surface than
if a natural surface is used. Cotton swabs may be used to control the first
more or less massive bleeding, followed by vigorous flushing with fixative.
Avoid the use of salt solutions for this purpose. In general, success with
this method will depend upon anatomically avoiding the larger blood
vessels so that massive hemorrhages do not occur.
Once again it is pertinent to stress that the eyes must be protected
from the fumes of osmium tetroxide, for under the pressure of working
efficiently and fast, it is all too easy to forget about one's eyes until
corneal damage has been done.
2.6. Vascular Perfusion
It is natural for one acquainted with the techniques of conventional
microscopy to think of vascular perfusion as a likely way of preserving
tissue for electron microscopy. This can be done very successfully when
buffered aldehyde fixatives are used, but cannot be done without heroic
means and extraordinary attention to detail with osmium tetroxide
fixation.
We have had a good deal of experience using buffered formaldehyde
(Chapters 3.9 and 3.10) as a perfusing fluid. Ordinarily we have used it as a
killing agent and primary fixative to gain time to retrieve deeply placed
tissue, in order to do careful dissections and to expose anatomically difficult
tissue, which then was fixed secondarily with osmium tetroxide. It is
ideal for this purpose, and makes it possible to collect well-fixed tissue
that would be difficult indeed to obtain in any other way. Furthermore, it
can be used to harden particularly soft tissues so that suitably small
blocks then can be prepared without mechanical damage for secondary
fixation with osmium tetroxide.
Our perfusion apparatus is commonplace and simple. It relies upon
gravity flow from a fixative bottle about 4 ft above the operating level.
The tube from this usually terminates in a 3-way stopcock of the type
used with syringe needles. Very large caliber syringe needles (for rats, 16
gage; for cats, 12 gage) then serve as cannulae. These usually are deliberately blunted, particularly when the perfusion is expected to be
directly into a heart ventricle. This whole system is filled with fixative in