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Sample Pr eparation of Cells and T issue 63

In the case of snap-freezing, the water contained within the cells
acts as the supporting medium. Fresh tissue is snap-frozen in liquid-
nitrogen-cooled isopentane (–170ºC) to promote vitreous ice formation
and to prevent ice-crystal damage, since the latter can produce holes in
the tissue and destroy cellular morphology and tissue architecture. The
hardened tissue can then be embedded in mounting medium such as
optimal cutting temperature (OCT) compound for sectioning within a
cryostat maintained at –17ºC and then subsequently stained.
OCT is a viscous solution at room temperature, consisting of a resin-
polyvinyl alcohol, an antifungal agent, benzalkonium chloride and
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polyethylene glycol to lower the freezing temperature. Turbett and
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Sellmer report that it is not advisable to store tissues in OCT for long
durations, since it was found that amplification of DNA, extracted from
these tissues, was significantly effected for segments of greater than 300
base pairs. However, RNA was found to be unaffected. It was suggested
that snap-frozen tissues should be stored without any medium.
Although the methods outlined above represent the mainstay
tissue processing techniques for paraffin embodiment/cryopreser-
vation in the present pathology laboratories, some researchers have
recently reported alternative tissue preparation protocols with the
view of optimizing the assessment of specific biomolecular domains.
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Gillespie conducted a comparative molecular profiling study in
clinical tissue specimens that were fixed for long-term storage with
widely used techniques (snap-frozen and formalin-fixed paraffin
embedded) and a less common method of 70 percent ethanol fixation
and paraffin embedding. The researchers found that although the
total protein quantity was decreased in fixed and embedded tissues
compared to snap-frozen tissue, 2D-PAGE analysis of proteins from
ethanol-fixed, paraffin-embedded prostate, shared 98 percent iden-
tity with a matched sample from the same patient that was snap-frozen,
indicating that the molecular weights and isoelectric points of the pro-
teins were not disturbed by the tissue-processing method. The gen-
eral quality and quantity of the proteins in the ethanol-fixed samples
were found to be superior to formalin-fixed tissue. Furthermore, Gil-
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lespie reports mRNA and DNA recovery were more pronounced in
ethanol-fixed specimens compared with formalin-fixed samples.
Thus, further improvements to tissue processing methodologies will
play a key role toward ultimately determining the complete molecu-
lar anatomy of normal and diseased human cell types.

3.2.2 Preparation of Tissues for Diagnostic Assessment
Using FTIR and Raman Microspectroscopy
Researchers working in the field of FTIR and Raman tissue diagnos-
tics have employed a variety of methods for tissue preparation. In the
first instance, FTIR spectroscopic studies have been carried out using
ground samples of snap-frozen tissue for the bulk analysis of chemical