3.10.
ALDEHYDES AS FIXATIVES FOR HISTOCHEMISTRY
57
histochemical reactions to be performed on otherwise completed sections.
In particular, specific enzymatic digestions often remained feasible. A
recent account of these efforts can be found in Leduc and Bernhard
(1962).
The recent comparative study of a number of aldehydes by Sabatini,
Bensch, and Barrnett (1963, 1964) would seem to indicate, however, that
formaldehyde is not the best aldehyde that may be used for histochemical
purposes. These investigators regard glutaraldehyde and acrylic aldehyde
(acrolein) as the most effective morphological fixatives when used as the
sole fixatives. Acrylic aldehyde will never be acceptable in most laboratories, though, for it is a violent tear gas, promoting intolerable lachrymation unless used in an unusually effective hood. Also, Luft (1959) pointed
out that solutions of this aldehyde are unstable and must be made up
fresh every few days, thus complicating the problems of handling it.
Fortunately, glutaraldehyde has a mild and fairly pleasant odor reminding one of a cider press. This is an interesting molecule, consisting of a
5-carbon chain with aldehyde groups on both ends. Thus it presumably
is capable of cross-linking compounds with which it might react. Quite
likely this contributes to its excellent qualities as a protein fixative, and
gives it an advantage over formaldehyde, even though its size is several
times that of the latter. Figure 4 is an example of tissue fixed in glutaraldehyde.
Sabatini, Bensch, and Barrnett (1963, 1964) considered glyoxal, crotonaldehyde, and methacrolein next in effectiveness, and then formaldehyde
[although as noted above (Chapter 3.9), I question the lowly status they
assigned to formaldehyde]. Decidedly inferior in preserving cytological
morphology was hydroxyadipaldehyde, but this compound has a special
interest histochemically as will become apparent below. Unsatisfactory
were pyruvic aldehyde and acetaldehyde. All of these various aldehydes
were compared when made up in phosphate or cacodylate buffers. The
buffer strength ordinarily was 0.1 M, but occasionally it was 0.2 M.
Small quantities of sucrose were added to control molarity, and varying
concentrations of the aldehydes were chosen. Whenever possible the pH
was adjusted so that it was slightly on the alkaline side. The account
which follows will speak particularly of glutaraldehyde and hydroxyadipaldehyde.
Sabatini and his co-workers recommend using glutaraldehyde either as
a 4 or a 6.5% solution in a 0.1 M phosphate or cacodylate buffer without
the addition of sugar. Normally, they fix from 0.5 to 2 hours. The tissue
ALDEHYDES AS FIXATIVES FOR HISTOCHEMISTRY
57
histochemical reactions to be performed on otherwise completed sections.
In particular, specific enzymatic digestions often remained feasible. A
recent account of these efforts can be found in Leduc and Bernhard
(1962).
The recent comparative study of a number of aldehydes by Sabatini,
Bensch, and Barrnett (1963, 1964) would seem to indicate, however, that
formaldehyde is not the best aldehyde that may be used for histochemical
purposes. These investigators regard glutaraldehyde and acrylic aldehyde
(acrolein) as the most effective morphological fixatives when used as the
sole fixatives. Acrylic aldehyde will never be acceptable in most laboratories, though, for it is a violent tear gas, promoting intolerable lachrymation unless used in an unusually effective hood. Also, Luft (1959) pointed
out that solutions of this aldehyde are unstable and must be made up
fresh every few days, thus complicating the problems of handling it.
Fortunately, glutaraldehyde has a mild and fairly pleasant odor reminding one of a cider press. This is an interesting molecule, consisting of a
5-carbon chain with aldehyde groups on both ends. Thus it presumably
is capable of cross-linking compounds with which it might react. Quite
likely this contributes to its excellent qualities as a protein fixative, and
gives it an advantage over formaldehyde, even though its size is several
times that of the latter. Figure 4 is an example of tissue fixed in glutaraldehyde.
Sabatini, Bensch, and Barrnett (1963, 1964) considered glyoxal, crotonaldehyde, and methacrolein next in effectiveness, and then formaldehyde
[although as noted above (Chapter 3.9), I question the lowly status they
assigned to formaldehyde]. Decidedly inferior in preserving cytological
morphology was hydroxyadipaldehyde, but this compound has a special
interest histochemically as will become apparent below. Unsatisfactory
were pyruvic aldehyde and acetaldehyde. All of these various aldehydes
were compared when made up in phosphate or cacodylate buffers. The
buffer strength ordinarily was 0.1 M, but occasionally it was 0.2 M.
Small quantities of sucrose were added to control molarity, and varying
concentrations of the aldehydes were chosen. Whenever possible the pH
was adjusted so that it was slightly on the alkaline side. The account
which follows will speak particularly of glutaraldehyde and hydroxyadipaldehyde.
Sabatini and his co-workers recommend using glutaraldehyde either as
a 4 or a 6.5% solution in a 0.1 M phosphate or cacodylate buffer without
the addition of sugar. Normally, they fix from 0.5 to 2 hours. The tissue