56 PART III Sample Preparation in Light Microscopy
buffered formaldehyde or glutardialdehyde fixative for up to 12 hours. You probably
know this as formalin. Fixative agent and the duration of fixation is crucial
and mainly depends on the size of the sample. Fixation is crucial as it prevents
decay and preserve cells and tissues in a »life-like« state by stopping enzyme activities.
It stabilizes the chemical components and hardens the specimen in anticipation
of further microscopic examination. An alternative approach for animal
tissues is to pass the fixative through the blood stream of the animal before removing
the organs. This technique called perfusion may help reduce artifacts, false
or inaccurate representations of the specimen that result from chemical treatment
or handling of the cells or tissues.
Cut Slices Thin Like Parma Ham
Once fixed, samples are placed into an embedding medium that will hold it rigidly
in position while sections are cut. Usually this is paraffin wax, but other materials
such as a glycerol-based freezing medium are also used to surround the
sample during sectioning. Sectioning then takes place on a microtome – a tool
comparable to a butcher’s meat slicer, which shaves the sample into thin slices
usually a few micrometers thick. As paraffin is insoluble in water, any water in the
specimen must first be removed by dehydration in alcohol and replaced by an organic
solvent such as xylene. Once cut, sections are mounted on a glass slide and
stained to bring out specific features and color characteristics of the sample before
being imaged on a microscope.
Bringing More Color into Life
Cells and other structures are colorless. If you want to reveal structural details
with brightfield microscopes you need to make them somehow visible. How? Try
some form of staining … Generally, you mount the fixed tissue on a microscope
slide and then treat it with any of a variety of dyes and stains that have been adapted
for this purpose. Sometimes the tissue is treated with a single stain, but more
often a series of stains is used, each with an affinity for a different kind of cellular
component. Read more about this in the next chapter!