36
3.
FIXATION
aldehyde fixation. Their use is contraindicated, however, if histochemical
considerations are involved. The beginner should by all means successfully accomplish what is possible by these means before turning to other
preservatives. He will not be seriously handicapping himself if he begins
with the original formula proposed by Palade in 1952. This simply uses
a rather strong buffering solution with osmium tetroxide added. The
buffer chosen was the acetate-Veronal buffer of Michaelis. Apparently
Palade empirically found this to be superior to inorganic buffers, and
there was the further theoretical consideration that the bivalent ions of
tissues would not form insoluble precipitates with it.
The fixative is prepared as follows:
Stock buffer solution:
Sodium Veronal (barbital)
14.7 gm
Sodium acetate
9.7 gm
Distilled water to make
500
ml
Stock osmium tetroxide solution:
Osmium tetroxide*
2%
Fixative:
Buffer
5
ml
O.lATHClf
5
ml
Distilled water
2.5 ml
2% osmium tetroxide solution
12.5 ml
The fixative should have a pH of about 7.4. Small corrections are made
by a few additional drops of buffer or acid as required before the distilled
water is added to make the 25 ml.
Both the buffer and the fixative can be kept a reasonable length of
* Special precaution must be observed in preparing osmuim tetroxide solutions. The
operator's respiratory tract, and particularly his eyes, must be protected from the fumes
when the ampule, in which it is packaged, is broken. It is best to smash this under
water in the container which is to be used for storage.
It is also essential that all glassware be free of organic contaminants for these will
combine with, and destroy, the osmium tetroxide. For this reason only glass-stoppered
bottles should be used. And for this reason, before breaking the ampule, its label must
be soaked off, and all traces of glue removed.
Osmium tetroxide dissolves very slowly, so that solutions containing it must be made
up at least one day before their intended use.
f One can make a 0.1 N HC1 solution by diluting 8.6 ml of concentrated HC1 (36%,
11.6 M) in water to make 1 liter.
3.
FIXATION
aldehyde fixation. Their use is contraindicated, however, if histochemical
considerations are involved. The beginner should by all means successfully accomplish what is possible by these means before turning to other
preservatives. He will not be seriously handicapping himself if he begins
with the original formula proposed by Palade in 1952. This simply uses
a rather strong buffering solution with osmium tetroxide added. The
buffer chosen was the acetate-Veronal buffer of Michaelis. Apparently
Palade empirically found this to be superior to inorganic buffers, and
there was the further theoretical consideration that the bivalent ions of
tissues would not form insoluble precipitates with it.
The fixative is prepared as follows:
Stock buffer solution:
Sodium Veronal (barbital)
14.7 gm
Sodium acetate
9.7 gm
Distilled water to make
500
ml
Stock osmium tetroxide solution:
Osmium tetroxide*
2%
Fixative:
Buffer
5
ml
O.lATHClf
5
ml
Distilled water
2.5 ml
2% osmium tetroxide solution
12.5 ml
The fixative should have a pH of about 7.4. Small corrections are made
by a few additional drops of buffer or acid as required before the distilled
water is added to make the 25 ml.
Both the buffer and the fixative can be kept a reasonable length of
* Special precaution must be observed in preparing osmuim tetroxide solutions. The
operator's respiratory tract, and particularly his eyes, must be protected from the fumes
when the ampule, in which it is packaged, is broken. It is best to smash this under
water in the container which is to be used for storage.
It is also essential that all glassware be free of organic contaminants for these will
combine with, and destroy, the osmium tetroxide. For this reason only glass-stoppered
bottles should be used. And for this reason, before breaking the ampule, its label must
be soaked off, and all traces of glue removed.
Osmium tetroxide dissolves very slowly, so that solutions containing it must be made
up at least one day before their intended use.
f One can make a 0.1 N HC1 solution by diluting 8.6 ml of concentrated HC1 (36%,
11.6 M) in water to make 1 liter.