234
7.
"STAINING"
acetate was used as the primary stain. Relatively short exposures of no
more than 30 minutes to a 5% solution are enough to have a dramatic
effect upon subsequent lead staining even when the embedment has been
an epoxy resin. After removing floating grids from drops of the uranyl
acetate staining solution, of course they must be washed briefly by floating
them on distilled water before staining with lead hydroxide. Then an exposure of no more than 15 minutes to Reynold's lead citrate stain can be
expected to yield an intense reaction. Figures 28(c) and (d) and 40 illustrate the contrast that can be achieved with double staining.
7.4. Uranyl Stains
The uranium salt which has proved to be most effective as a staining
agent is uranyl acetate. Watson (1958) appears to have been the first
investigator to explore its use as an electron stain. He originally regarded
this as being a stain with little specificity, and it is so used commonly
today. As a general purpose stain uranyl acetate probably should be
regarded as superior to phosphotungstic acid, but inferior to the lead
stains. Proteins are apt to be stained fairly intensely. However, it does
not stain cytomembranes particularly vividly, and so does not produce
the dramatic pictures that the lead stains are capable of [compare Fig.
28(b) with 28(a)].
Recently Huxley and Zubay (1961) have demonstrated that DNA binds
a specific amount of uranyl acetate (approximately equivalent to its own
FIG. 28. Sections of rat liver from the same block stained in different ways. Liver
fixed with buffered formaldehyde followed by Millonig's osmium tetroxide. Embedded
in cross-linked methacrylate.
(a) Unsupported section, stained with Watson's lead hydroxide. Note chromatin,
ribosomes, and cytomembranes vividly stained. Mitochondrial matrix and diffuse cytoplasmic protein weakly stained.
(b) Unsupported section, stained with uranyl acetate. Note mitochondrial matrix and
diffuse cytoplasmic protein more darkly stained in comparison with nucleoproteins and
cytomembranes, giving less over-all contrast than with lead hydroxide (a).
(c) Unsupported section, stained first with uranyl acetate and then lead hydroxide.
Extreme contrast results, with diffuse protein and mitochondrial matrix being stained
vigorously as well as nucleoproteins and cytomembranes.
(d) Section sandwiched with evaporated carbon layers after staining first with uranyl
acetate, and then lead hydroxide. All components shown with maximum detail evident.
Extreme contrast of (c) reduced.
7.
"STAINING"
acetate was used as the primary stain. Relatively short exposures of no
more than 30 minutes to a 5% solution are enough to have a dramatic
effect upon subsequent lead staining even when the embedment has been
an epoxy resin. After removing floating grids from drops of the uranyl
acetate staining solution, of course they must be washed briefly by floating
them on distilled water before staining with lead hydroxide. Then an exposure of no more than 15 minutes to Reynold's lead citrate stain can be
expected to yield an intense reaction. Figures 28(c) and (d) and 40 illustrate the contrast that can be achieved with double staining.
7.4. Uranyl Stains
The uranium salt which has proved to be most effective as a staining
agent is uranyl acetate. Watson (1958) appears to have been the first
investigator to explore its use as an electron stain. He originally regarded
this as being a stain with little specificity, and it is so used commonly
today. As a general purpose stain uranyl acetate probably should be
regarded as superior to phosphotungstic acid, but inferior to the lead
stains. Proteins are apt to be stained fairly intensely. However, it does
not stain cytomembranes particularly vividly, and so does not produce
the dramatic pictures that the lead stains are capable of [compare Fig.
28(b) with 28(a)].
Recently Huxley and Zubay (1961) have demonstrated that DNA binds
a specific amount of uranyl acetate (approximately equivalent to its own
FIG. 28. Sections of rat liver from the same block stained in different ways. Liver
fixed with buffered formaldehyde followed by Millonig's osmium tetroxide. Embedded
in cross-linked methacrylate.
(a) Unsupported section, stained with Watson's lead hydroxide. Note chromatin,
ribosomes, and cytomembranes vividly stained. Mitochondrial matrix and diffuse cytoplasmic protein weakly stained.
(b) Unsupported section, stained with uranyl acetate. Note mitochondrial matrix and
diffuse cytoplasmic protein more darkly stained in comparison with nucleoproteins and
cytomembranes, giving less over-all contrast than with lead hydroxide (a).
(c) Unsupported section, stained first with uranyl acetate and then lead hydroxide.
Extreme contrast results, with diffuse protein and mitochondrial matrix being stained
vigorously as well as nucleoproteins and cytomembranes.
(d) Section sandwiched with evaporated carbon layers after staining first with uranyl
acetate, and then lead hydroxide. All components shown with maximum detail evident.
Extreme contrast of (c) reduced.