50
3.
FIXATION
3.8
Aldehyde Fixation: Introductory Remarks
The properties of formalin have tempted a number of electron microscopists to explore its use. However, it soon became apparent that formaldehyde-fixed tissue was particularly prone to "explosion damage" when
methacrylate was the embedment. Such tissue also proved to be unduly
sensitive to electron bombardment, and sometimes decomposed as it was
being observed (Morgan et al., 1956). However, these effects could be
minimized if a primary fixation with formalin was followed by a secondary treatment with an osmium tetroxide fixative. It was realized early
that some botanical material having heavy walls was particularly difficult
to preserve with osmium tetroxide for the fixative often did not penetrate.
Formalin sometimes proved to be effective under these circumstances, and
Ehrlich (1958) fairly successfully used neutralized formalin followed by
Palade fixative even with microspores and pollen grains.
There are, of course, many histochemical reactions which can be performed on tissue after an aldehyde fixation, but which will not work
following osmium tetroxide. Thus, histochemists soon started using a
primary fixation with formalin, which was then followed by enzymatic
digestions or other histochemical reactions, and finally a secondary fixation with osmium tetroxide. In this manner, Holt and Hicks (1961) were
able to visualize nicely the localization of acid phosphatase, and thus
demonstrate lysosomes in liver sections. The quality of the control specimens made it evident that a primary fixation with formaldehyde did
little or no harm as far as final pictures of cell organelles were concerned.
The material looked almost as though it had been preserved originally
with osmium tetroxide. Luft (1959) had demonstrated many of the same
virtues in acrylic aldehyde (acrolein) when used as a primary
fixative.
Indeed, he preferred this to formaldehyde, but for reasons that no longer
seem valid.
One reason for the considerable success of Holt and Hicks (1961) was
their deliberate choice of a phosphate buffer for their formaldehyde. They
did not visualize the full potentiality of the method, however, for they
limited themselves to methacrylate embedding procedures without any
particular precautions to prevent electron beam damage (see Chapter 4.9).
It was not possible to assess the true virtues of formalin fixation until it
was used with a phosphate buffer in conjunction with an embedment in
a cross-linked plastic as indicated by Pease (1962). He demonstrated that
3.
FIXATION
3.8
Aldehyde Fixation: Introductory Remarks
The properties of formalin have tempted a number of electron microscopists to explore its use. However, it soon became apparent that formaldehyde-fixed tissue was particularly prone to "explosion damage" when
methacrylate was the embedment. Such tissue also proved to be unduly
sensitive to electron bombardment, and sometimes decomposed as it was
being observed (Morgan et al., 1956). However, these effects could be
minimized if a primary fixation with formalin was followed by a secondary treatment with an osmium tetroxide fixative. It was realized early
that some botanical material having heavy walls was particularly difficult
to preserve with osmium tetroxide for the fixative often did not penetrate.
Formalin sometimes proved to be effective under these circumstances, and
Ehrlich (1958) fairly successfully used neutralized formalin followed by
Palade fixative even with microspores and pollen grains.
There are, of course, many histochemical reactions which can be performed on tissue after an aldehyde fixation, but which will not work
following osmium tetroxide. Thus, histochemists soon started using a
primary fixation with formalin, which was then followed by enzymatic
digestions or other histochemical reactions, and finally a secondary fixation with osmium tetroxide. In this manner, Holt and Hicks (1961) were
able to visualize nicely the localization of acid phosphatase, and thus
demonstrate lysosomes in liver sections. The quality of the control specimens made it evident that a primary fixation with formaldehyde did
little or no harm as far as final pictures of cell organelles were concerned.
The material looked almost as though it had been preserved originally
with osmium tetroxide. Luft (1959) had demonstrated many of the same
virtues in acrylic aldehyde (acrolein) when used as a primary
fixative.
Indeed, he preferred this to formaldehyde, but for reasons that no longer
seem valid.
One reason for the considerable success of Holt and Hicks (1961) was
their deliberate choice of a phosphate buffer for their formaldehyde. They
did not visualize the full potentiality of the method, however, for they
limited themselves to methacrylate embedding procedures without any
particular precautions to prevent electron beam damage (see Chapter 4.9).
It was not possible to assess the true virtues of formalin fixation until it
was used with a phosphate buffer in conjunction with an embedment in
a cross-linked plastic as indicated by Pease (1962). He demonstrated that