Determination of Altered Mitochondria Ultrastructure by Electron Microscopy - Protein Misfolding and Cellular Stress in Disease and Aging - page 271

The temperature of fixation can occasionally be critical. For
example, certain labile structures, such as certain microtubules,
may be lost as a result of rearrangement when glutaraldehyde
is used in a cold environment. The most effective means of
minimizing the deterioration of purified glutaraldehyde is by
storing as an unbuffered, 10-25% solution at subfreezing
temperatures (~ −20°C). It is advisable not to mix the buffer
and the fixative until immediately before use. Less polymer-
ization occurs when glutaraldehyde is stored in a cacodylate
buffer than when it is stored in a phosphate buffer at the same
pH (7.4).
2. As cacodylate buffer contains toxic arsenic, careful attention
should be paid to the management of the reagent and the
treatment of discharge. Cacodylate buffer is quite effective
within a pH range of ~6.4-7.4.
3. One well-known example is the so-called “tooth-paste” arti-
fact of the spinal cord in which a portion of tissue is con-
stricted leading to the abnormal displacement of tissue above
and below the constriction. Regions of interest (e. g., infarcts,
tumors) are especially prone to such changes.
4. Sections should be as thin as possible, i.e. <1 mm thick.
5. The nervous system is particularly vulnerable to postmortem
delay. Postmortem autolysis is a common artifact resulting
from delayed fixation. Speedy removal of the tissue samples of
the nervous system in particular is important, as is the prompt
fixation of specimens, in order to minimize the possibility
of any delayed-fixation artifacts. In animal experiments,
intracardial or intravenous perfusion with heparinized saline
(pH 7.4) is performed to achieve suitable fixation, followed
by perfusion with ice-cold 4% paraformaldehyde and 0.2%
glutaraldehyde in 0.1 M phosphate buffer (pH 7.4).
6. Sectioned tissues should be as thin as possible (i.e. at least
<1 mm thick) and when necessary, as large as possible (i.e. a
5 mm square from the cerebral cortex, or a large enough
sample from the spinal cord to include the entire anterior
horn to facilitate anatomical orientation). Sectioned tissue
can then be trimmed to the appropriate size upon semi-thin
tissue sectioning.
7. Higher concentrations of osmium tetroxide exhibit good
penetration into tissues. The degree of tissue penetration is
the same at temperature, ranging between at 4°C and room
temperature, but with increasing temperature, tissue penetra-
tion inversely diminishes. Currently, the most commonly used
concentrations of osmium tetroxide range from 1 to 2% in
buffer. The procedure involving osmium tetroxide should be
carried out in a draft chamber.