3.10.
ALDEHYDES AS FIXATIVES FOR HISTOCHEMISTRY
59
minutes) exposures to the aldehydes when post-fixation with osmium
tetroxide was contemplated, but given long exposures (2-4 hours) when
used as the only fixative. However, I am willing to defer to the much
larger experience that other investigators have had which does seem to
favor glutaraldehyde as the fixative of choice when this is to be the sole
preservative agent.
The cliche "you cannot have your cake and eat it too" rarely is more
apt than in considering the problem of retaining excellent cytological
morphology while at the same time keeping enzyme systems intact.
Sabatini et al. have demonstrated that there is an almost inverse relationship in the effectiveness of the different aldehydes in these two regards.
Hydroxyadipaldehyde proved to be by far the most effective fixing agent
in retaining residual enzymatic activity, although its properties as a
morphological fixative were poor. Thus tissue fixed in this agent retained
a high degree of succinic dehydrogenase, cytochrome oxidase (without
exogenous cytochrome), and glucose-6-phosphatase activity, while all of
the other tested aldehydes either destroyed these totally or, at best, left
only moderate activity quite different in intensity from that in hydroxyadipaldehyde-preserved tissue. Yet these authors found curious exceptions
so that no fully satisfactory generalization is possible. Thus, hydroxyadipaldehyde-preserved tissue showed only traces of cytoplasmic esterase
activity which was commonly found in substantial amounts after other
aldehydes had been used. Actually, glutaraldehyde left intact at least
moderate responses to many reactions while also providing excellent
morphological preservation. In contrast, acrylic aldehyde destroyed almost
all enzymatic activity without producing a better morphological preservation than glutaraldehyde.
Sabatini et al. used hydroxyadipaldehyde in a 12.5% concentration,
dissolved in 0.1 M phosphate or cacodylate buffer, with sucrose added to
establish a 0.44 molarity. The pH was adjusted to 7.5. This writer has had
the opportunity to see a number of micrographs of tissue preserved in
this way in Dr. Barrnett's collection. There was no doubt that the tissue
was morphologically poorly preserved by any conventional standards of
judgment. However, ordinarily organelles could be identified in cells,
and of course the histochemical information obtained justified the means.
The writings of Sabatini and his co-workers emphasize that the
fixation
of tissue sometimes will have to be quite carefully tailored to the histochemical reactions that are to be performed. The interested histochemist,
of course, will wish to consult the original papers.
ALDEHYDES AS FIXATIVES FOR HISTOCHEMISTRY
59
minutes) exposures to the aldehydes when post-fixation with osmium
tetroxide was contemplated, but given long exposures (2-4 hours) when
used as the only fixative. However, I am willing to defer to the much
larger experience that other investigators have had which does seem to
favor glutaraldehyde as the fixative of choice when this is to be the sole
preservative agent.
The cliche "you cannot have your cake and eat it too" rarely is more
apt than in considering the problem of retaining excellent cytological
morphology while at the same time keeping enzyme systems intact.
Sabatini et al. have demonstrated that there is an almost inverse relationship in the effectiveness of the different aldehydes in these two regards.
Hydroxyadipaldehyde proved to be by far the most effective fixing agent
in retaining residual enzymatic activity, although its properties as a
morphological fixative were poor. Thus tissue fixed in this agent retained
a high degree of succinic dehydrogenase, cytochrome oxidase (without
exogenous cytochrome), and glucose-6-phosphatase activity, while all of
the other tested aldehydes either destroyed these totally or, at best, left
only moderate activity quite different in intensity from that in hydroxyadipaldehyde-preserved tissue. Yet these authors found curious exceptions
so that no fully satisfactory generalization is possible. Thus, hydroxyadipaldehyde-preserved tissue showed only traces of cytoplasmic esterase
activity which was commonly found in substantial amounts after other
aldehydes had been used. Actually, glutaraldehyde left intact at least
moderate responses to many reactions while also providing excellent
morphological preservation. In contrast, acrylic aldehyde destroyed almost
all enzymatic activity without producing a better morphological preservation than glutaraldehyde.
Sabatini et al. used hydroxyadipaldehyde in a 12.5% concentration,
dissolved in 0.1 M phosphate or cacodylate buffer, with sucrose added to
establish a 0.44 molarity. The pH was adjusted to 7.5. This writer has had
the opportunity to see a number of micrographs of tissue preserved in
this way in Dr. Barrnett's collection. There was no doubt that the tissue
was morphologically poorly preserved by any conventional standards of
judgment. However, ordinarily organelles could be identified in cells,
and of course the histochemical information obtained justified the means.
The writings of Sabatini and his co-workers emphasize that the
fixation
of tissue sometimes will have to be quite carefully tailored to the histochemical reactions that are to be performed. The interested histochemist,
of course, will wish to consult the original papers.