3.9.
BUFFERED ALDEHYDES AS PRIMARY
FIXATIVES
55
In speaking of phosphate buffers, one normally thinks of Sorensen's
in which suitable and equal molar concentrations of monobasic and
dibasic phosphates are blended appropriately. If 12 parts of monobasic
solution are blended with 38 parts of dibasic solution, a pH of 7.2 should
result. Changing the ratio to 9:32 should produce a pH of 7.4.
Sodium cacodylate is an organic arsenical salt with buffering properties
in the lower alkaline range. A 0.1 M solution (21.4 gm/liter) is adjusted
to pH 7.2-7.4 by the addition of drops of concentrated HC1. Sodium
cacodylate is available from the Amend Drug and Chemical Co., New
York. For further information on the preparation of buffers, Clark (1928)
should be consulted.
In our own laboratory we have been interested in formaldehyde as a
"killing agent" and a primary fixative before osmium tetroxide fixation.
As such, we routinely perfuse it into animals via the arterial system
(Chapter 2.6), or we cut free-hand slices of tissues as thin as possible, and
treat them first with formaldehyde (Chapter 2.3). We expect the formaldehyde to penetrate rapidly and block autolysis. We gain time to dissect
carefully to find deep structures, to collect a variety of tissues, and to
deal with tissues that require some hardening before they can be diced
properly for osmium tetroxide fixation. In using formaldehyde in this
way, usually it has been possible to pass suitably small pieces of tissue on
into Millonig's osmium tetroxide fixative within 10 minutes. When complex dissections have been necessary, however, there may be delays of a
half hour or more. Indeed, the experience of Sabatini, Bensch, and
Barrnett (1963) indicates that tissue can be kept almost indefinitely in
aldehyde solutions without deterioration, or even in suitably buffered
media following aldehyde fixation.
Figures 3, 10, 12, 28, 39, and 40 illustrate what may be expected from
these procedures. Figure 3 is of retinal material taken from a cat, the
whole fore end of which had been perfused via the aorta. Blanching of
the eyes was noted as the fixative reached the head. Eye material, however, was not removed until after about 30 minutes had elapsed. It was
only then that small sheets of tissue were treated with osmium tetroxide.
Nonetheless, subsequent study demonstrated that all parts of the retina
FIG. 3. Base of cat retina including outer rod segments, unpigmented pigment cells,
and adjoining choroidal layers including capillaries and tapetum. Fixed by perfusion
with buffered formaldehyde and subsequently treated with Millonig's buffered osmium
tetroxide. Embedded in Araldite, stained with Watson's lead hydroxide. Note that
the various cell layers remained in place during the processing of the tissue. The
detail at the upper left demonstrates the preservation of mitochondrial and outer rod
segment membrane systems at fairly high magnification.
BUFFERED ALDEHYDES AS PRIMARY
FIXATIVES
55
In speaking of phosphate buffers, one normally thinks of Sorensen's
in which suitable and equal molar concentrations of monobasic and
dibasic phosphates are blended appropriately. If 12 parts of monobasic
solution are blended with 38 parts of dibasic solution, a pH of 7.2 should
result. Changing the ratio to 9:32 should produce a pH of 7.4.
Sodium cacodylate is an organic arsenical salt with buffering properties
in the lower alkaline range. A 0.1 M solution (21.4 gm/liter) is adjusted
to pH 7.2-7.4 by the addition of drops of concentrated HC1. Sodium
cacodylate is available from the Amend Drug and Chemical Co., New
York. For further information on the preparation of buffers, Clark (1928)
should be consulted.
In our own laboratory we have been interested in formaldehyde as a
"killing agent" and a primary fixative before osmium tetroxide fixation.
As such, we routinely perfuse it into animals via the arterial system
(Chapter 2.6), or we cut free-hand slices of tissues as thin as possible, and
treat them first with formaldehyde (Chapter 2.3). We expect the formaldehyde to penetrate rapidly and block autolysis. We gain time to dissect
carefully to find deep structures, to collect a variety of tissues, and to
deal with tissues that require some hardening before they can be diced
properly for osmium tetroxide fixation. In using formaldehyde in this
way, usually it has been possible to pass suitably small pieces of tissue on
into Millonig's osmium tetroxide fixative within 10 minutes. When complex dissections have been necessary, however, there may be delays of a
half hour or more. Indeed, the experience of Sabatini, Bensch, and
Barrnett (1963) indicates that tissue can be kept almost indefinitely in
aldehyde solutions without deterioration, or even in suitably buffered
media following aldehyde fixation.
Figures 3, 10, 12, 28, 39, and 40 illustrate what may be expected from
these procedures. Figure 3 is of retinal material taken from a cat, the
whole fore end of which had been perfused via the aorta. Blanching of
the eyes was noted as the fixative reached the head. Eye material, however, was not removed until after about 30 minutes had elapsed. It was
only then that small sheets of tissue were treated with osmium tetroxide.
Nonetheless, subsequent study demonstrated that all parts of the retina
FIG. 3. Base of cat retina including outer rod segments, unpigmented pigment cells,
and adjoining choroidal layers including capillaries and tapetum. Fixed by perfusion
with buffered formaldehyde and subsequently treated with Millonig's buffered osmium
tetroxide. Embedded in Araldite, stained with Watson's lead hydroxide. Note that
the various cell layers remained in place during the processing of the tissue. The
detail at the upper left demonstrates the preservation of mitochondrial and outer rod
segment membrane systems at fairly high magnification.