Embedding tissues to make paraffin blocks is now almost a reference standard in diagnostic histology. It can be done relatively fast and enables comparatively thin sections to be cut with good definition. Overall, preparation involves dehydration, clearing, infiltration, and processing.
Dehydration
Once the piece of tissue has been fixed it is necessary to remove all of its water, a step referred to as “dehydration”. The reasons for this are quite straightforward. The final goal is to have a piece of tissue with paraffin wax intimately surrounding the proteins, and paraffin wax and water do not mix in any proportion, so one of them has to go. That one is the water.
Note that it is not just a case of having the tissue encased in paraffin wax, the proteins themselves have to be both permeated with, and surrounded by, the wax. Wherever there were water molecules in the original fresh, unfixed tissue there must now be hardened paraffin wax, right down to the subcellular level.
Clearing
Most clearants (clearing agents) are hydrocarbons of one kind or another. Many clearants are inflammable and this is a serious concern. The saying that “familiarity breeds contempt” is valid, and many technologists treat clearing agents as if they are as safe as water. They are not. Always keep in mind that using such fluids on an electric machine with the possibility of sparking is inherently unsafe, especially when the fluids are heated to assist with their action. Fire blankets and appropriate fire extinguishers must be available in a place where they can be easily accessed should a fire begin. Do not let this give a false sense of security though, and if the fire is beyond simple measures to extinguish, leave and call the fire department. Do not put your life in jeopardy.
As with dehydration there is some shrinkage during clearing, but it is not excessive and varies according to the particular fluid used.
The derivation of the term “clearing” is explained in two different ways. One view is that the term refers to the dehydrant being “cleared” (i.e. removed) from the tissue. The other view is that it refers to making the tissue “clear” (i.e. translucent). Another term is occasionally encountered because of the latter view, “wax antemedium”, which refers to any fluid used immediately prior to infiltration. Most technologists do not differentiate between the two synonyms.
Infiltration
Paraffin wax is the most commonly used wax for infiltration during tissue preparation. This is composed of straight chain or n-alkanes with a carbon chain length of between 20 and 40. It is solid at room temperature but melts at temperatures up to about 65°C or 70°C. It is invariably a mixture, so the melting point is not sharp. It is inflammable, but not dangerously explosive, and is the wax used to make common candles. Normal precautions make it reasonably safe to use.
Paraffin wax can be purchased with melting points at different temperatures, the most common for histological use being about 56°C–58°C, although a somewhat higher temperature may be used in tropical countries, and a slightly lower for very delicate tissues. At its melting point it tends to be slightly viscous, but this decreases as the temperature is increased. The traditional advice is that it should be used at about 2°C above its melting point, but technologists often increase the temperature to above 60°C or 65°C in practice to decrease viscosity. This does cause some extra damage to the tissue, but facilitates more thorough infiltration.
It should be melted as required rather than kept in the molten state for weeks. Hot molten wax slowly deteriorates and eventually develops a greasy feel and may be discolored. It is then unsuitable for infiltration. In normal use this is not a serious concern, but in the past, when manual processing was the norm, it was common practice to keep a container of molten wax in an oven for months at a time and this would eventually deteriorate, particularly if stored at elevated temperatures.
Processing
A number of options exist for paraffin wax processing:
