Page 86 - Vibrational Spectroscopic Imaging for Biomedical Applications

62 Cha pte r T h ree

Displacement of water with ethanol preserves the secondary structures
of proteins, however, denatures their tertiary structure. Furthermore,
formalin or ethanol induces coagulation of the globular proteins
present in the cytoplasm, which can result in the loss of structural
integrity of organelles such as mitochondria. Another disadvan-
tage is that ethanol precipitates lipid molecules that are not pre-
served through the primary fixation step. However, stabilization of
intercellular proteins by formalin and ethanol localizes associated
glycogen.
Following dehydration, the alcohol is replaced by an organic sol-
vent such as xylene, which is miscible with both alcohol and molten
paraffin wax. The specimen is then immersed in and permeated by
molten paraffin wax. The infiltration of the wax into the intracellular
spaces is promoted by the previous ethanol dehydration step that
created pores in the cell’s plasma membrane. The specimen is then
cooled to room temperature, which solidifies the wax. This process
provides a physical support to the sample enabling thin sections
(usually 2 to 7 μm) to be cut without deformation of the cellular struc-
ture or architecture.
It is important to note that the process of fixation is not instanta-
neous and two important properties of the fixative are its penetration
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rate and binding time. Medawar was the first to demonstrate that
fixatives obey the diffusion laws, whereby the depth of penetration
was proportional to the square root of time. The importance of fixa-
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tive binding time was highlighted by Fox et al. who investigated
the binding of formaldehyde to rat kidney tissue, in which 16-μm-
thin sections were used so that penetration would not be considered
a factor in the kinetics of the reaction. They found that the amount
of methylene glycol that covalently bound to this tissue increased
with time until equilibrium was reached at 24 hours. Thus, binding
time is the limiting factor for tissue stabilization. These aspects of
chemical fixation (penetration rate and binding time) may be a
potential source of biomolecular variance in pathological samples,
since there exists a time lag in fixative exposure and binding between
cells located within the core of the tissue compared with those at the
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extreme dimensions of the block. Infact, Fox et al. report that
cells at the periphery of the tissue exhibit different morphological
properties to cells that are a few tenths of a millimeter further within
the specimen.
For molecular-based studies, snap-freezing of fresh tissue is
generally preferred, since this method avoids the use of organic
solvents that cause degradation or loss of some cellular compo-
nents. In particular, frozen sections are used to study enzymes and
soluble lipids. Furthermore, this method is used to conduct immu-
nohistochemical analysis, since some antigens may be affected by
extensive cross-linking chemical fixatives that denature their ter-
tiary structure.